Analytical Letters, 41: 24–35, 2008

Copyright # Taylor & Francis Group, LLC

ISSN 0003-2719 print/1532-236X online
DOI: 10.1080/00032710701746873

CHEMICAL AND BIOSENSORS

A Sequence-Selective Electrochemical DNA

Biosensor Based on HRP-Labeled Probe for
Colorectal Cancer DNA Detection

Zhijie Wang,1 Yunhui Yang,2 Kailiang Leng,1 Jishan Li,2

Fan Zheng,2 Guoli Shen,2 and Ruqin Yu2
1
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery
Sciences, Qingdao
2
State Key Laboratory for Chemo/Biosensing and Cheometrics, College
of Chemistry and Chemical Engineering, Hunan University, Changsha,
China

Abstract: A highly-sensitive sequence-selective DNA sensor based on HRP-labeled

probe to detect specific K-ras gene which is highly associated with colorectal cancer
has been reported. Capture probe modified with– SH was first chemically adsorbed
on the gold electrode through self-assembly. Then, the hybridization of a complemen-
tary nucleic acid (target DNA:K-ras gene) and HRP labeled oligonucleotide detection
probe occurred in a sandwich way. Finally, H2O2 electroreduction current catalyzed by
HRP was measured amperometrically in the presence of hydroquinon as mediator. The
sequence selectivity is double guaranteed by the complementary hybridization of target
DNA with capture and detection probes. The experimental conditions were optimized.
The linear range is 1.17
10211  1.17
1027 mol l21 with a detection limit of
5.85
10212 mol l21. The electrode with capture probe can be reused after regener-
ation in boiling water.

Keywords: Electrochemical DNA biosensor, HRP-labeled probe, colorectal cancer,

self-assembly, sandwich

Received 17 September 2007; accepted 1 November 2007

Financial support from the National Natural Science Foundation of China (Grant
Nos. 20435010, 20375012, 20205005) is gratefully acknowledged.
Address correspondence to Zhijie Wang, Yellow Sea Fisheries Research Institute
Chinese Academy of Fishery Sciences, Qingdao 266071. E-mail: zhijiewang2004@
yahoo.com.cn

24
Electrochemical DNA Biosensor Based on HRP-labeled Probe 25

INTRODUCTION

Wide-scale genetic testing requires the development of easy-to-use, fast, inex-

pensive, miniaturized analytical devices to acquire genetic information for the
diagnosis of inherited diseases. Various techniques such as gel electrophoresis
(Vossen and Fried 1997; Izzo et al. 2000) and dot blots (Evans et al. 1998;
Zhang et al. 2001) have been developed for DNA detection. However, these
techniques are usually too slow and labor-intensive. Electrochemical DNA bio-
sensors have received considerable attention for the detection of DNA hybrid-
ization (Palecek et al. 1998; Cai et al. 2003; Wang et al. 2003; Xie et al. 2004;
Zhu et al. 2004). Such sensors rely on the immobilization of a single-stranded
(ss) oligonucleotide probe onto an electrochemical transducer surface to
recognize its complementary target sequence. The high sensitivity of such
devices, coupled to their compatibility with modern micro fabrication technol-
ogies, together with their portability, low cost (disposability) and minimal
power requirements, make them excellent candidates for DNA diagnostics
(Wang 2002). For the DNA hybridization studies, direct electrooxidation of
DNA belongs to the early used electrochemical method. Among the four
nucleic acid bases, the guanine moiety is most easily oxidized and is most
suitable for such indicator-free hybridization detection (Ihara et al. 1997;
Souteyrand et al. 1997; Wang et al. 1998; Abbaspour and Mehrgardi 2004).
However, these direct electrochemical DNA biosensors usually have relatively
low sensitivity. Other kind electrochemical DNA biosensors that depend on
electro-active indicators such as a cationic metal complex and intercalating
organic compound (Hashimoto and Ishmori 1994; Mikklesen 1996) to
indicate the hybridization have also been studied. Most of these kinds of elec-
trochemical DNA probes are based on the use of intercalators. As these agents
were just intercalated within the double helix of DNA during hybridization, the
non-covalent bond with DNA is relatively weak, limiting the sensitivity of the
detection. Enzyme-labeled detection DNA probe can avoid the nonspecific
adsorption. In addition, the labeling of the target DNA is not required, which is
favorable to their application to real systems. Lumley-Woodyear (DeLumley
et al. 1996) introduced a direct enzyme-amplified technology to detection DNA
hybridization with improved sensitivity by a relatively complex procedure of
attaching the probe DNA to a film of polyacrylamide-based containing
electron-conducting redox hydrogel.
The incidence of colorectal cancer is very high in west Europe, Australia
and New Zealand, ranked the second in visceral cancer. In the U.S.A, color-
ectal cancer represents 15% of all cancers and is the second cause of
cancer-related mortality (Cohen et al. 1993). In the Netherlands, in 1990, it
was the third most frequent cause of death in men and the second most
frequent in women. Approximately one in three colorectal cancer patients
die from the complications of unresectable primary disease (Kievet and
Brunvels 1995). So, searching a highly sensitive and selective biosensor for
early stage cancer diagnosis is of considerable interest.
26 Z. Wang et al.

In this paper, a high-sensitive DNA sensor is reported based on HRP-

labeled probe to detect specific sequence of K-ras gene which is highly associ-
ated with colorectal cancer. A capture probe (probe DNA) modified with–SH
was firstly chemically adsorbed on the gold electrode through self-assembly.
The target DNA which contains complementary sequence with the probe
DNA would be captured by it through hybridization. The HRP labeled oligonu-
cleotide (detection DNA) which is complementary with another part of target
DNA would be hybridization in a sandwich way. Finally, H2O2 electroreduction
current catalyzed by HRP was measured amperometrically in the presence of
hydroquinon as the mediator. The simple manipulation procedure, high selectiv-
ity, low-cost, fast response, and broad linear range are the main features of
proposed DNA sensor. Through removal of target DNA and detection DNA
in hot hybridization buffer, the sensor can be regenerated.

EXPERIMENTAL

Reagents and Solutions

The oligonucleotide sequences associated with colorectal cancer (Table 1)

used in this study were obtained from Dalian Biotechnology Co., Ltd.
(Dalian, China). The target DNA sequence is the gene sequence associated
with colorectal cancer. The probe DNA and detection DNA are the
sequence complementary with corresponding parts of the target DNA.
The mismatch DNA is shown with four-mismatched bases underlined. The
random DNA is non-complementary with capture and detection probes.
Hydrogen peroxide (30% v/v aqueous solution), hydroquinone, thioglycolic
acid and glutaraldehyde (25%) were purchased from Shanghai Chemical
Reagents (Shanghai, China). Lyophilized horseradish peroxidase (HRP,
EC 1. 11. 17, RZ . 3.0, A . 250 U mg21) was obtained from Shanghai Bio-
chemical Reagents (Shanghai, China). L-Lysine was supplied by Tianjin
Guangfu Fine Chemical Research Institute (Tianjin, China).

Table 1. Oligonucleotide sequences used in this work

Target DNA 50 -AAAACTTGTGGTAGTTGGAGCT GATGGCGTAGGCAA-

GAGTGCCC-30
Probe DNA 50 -CTCCAACTACCACAAGTTTT-(CH2)6-SH-30
Detection DNA 50 -NH2-(CH2)3-GGGCACTCTTGCCTACGCCA-30
Mismatch DNAa 50 -AAATCTTGTGGTAGTTGTAGCT GATGGCGCAGGCAA-
GAGTGCGC-30
Random DNA 50 -GGGCACTCTTGCCTACGCCATCAGCTCCAACTACCA-
CAAGTTTT-30
a
Mismach DNA. The lineate bases are mismatched ones.
Electrochemical DNA Biosensor Based on HRP-labeled Probe 27

Hybridization buffer solution was prepared with 0.05 mol l21 NaCl
0.01 mol l21 Tris 0.001 mol l21 EDTA (pH 8.8, STE) in asepsis water and
stored in a refrigeratory. The supporting electrolyte was 0.067 mol l21
phosphate buffer solution containing 0.3 mol l21 NaCl (pH 7.0, PBS). Other
chemicals were of analytical reagent grade. All solutions were prepared
with doubly distilled water.

Apparatus

Cyclic voltammetry and amperometric analysis were carried out using a CHI
760b Electrochemical Analyzer (Chen Hua Instrument Inc., Shanghai, China).
The three-electrode system consisted of gold electrode (AuE, 1 mm diameter)
as the working electrode, a SCE as the reference electrode and a Pt foil as the
counter electrode. Cyclic voltammetric experiments were performed in
unstirred solutions. Amperometric measurements were carried out in stirred
substrate solutions with a steady-state background current first obtained
before standard H2O2 solution was added into the buffer solution. All poten-
tials were measured and reported versus the SCE. A magnetic stirrer and bar
provided convective transport.

Crosslinking of Detection DNA with HRP

A reaction mixture was prepared by mixing 100 ml of detection DNA (72.5 m

mol l21), 100 ml of glutaraldehyde (2.5%) and 200 ml of HRP (10 mg ml21,
pH 7.00), and the reaction lasted for 7 h at 48C. Then 100 ml of L -Lysine
(0.1%) was added to block for 3 h at 48C. The reaction mixture was
dialyzed overnight against 0.1 mol l21 phosphate buffer (pH 7.00) at 48C to
remove the non-reacted low molecular reagents.

Preparation of the DNA Electrode

The AuE surface was treated before modification by polishing with 0.05 mm
alumina/water slurry on a polishing cloth. The electrodes were then soaked
for 30 min in a freshly prepared piranha solution (30% H2O2/70% concen-
trated H2SO4). Subsequently, the electrodes were cleaned by cycling
between the potentials 21.0 and þ1.0 V versus SCE in 0.1 mol l21 H2SO4
solution at a scan rate of 100 mV/s for approximately 20 min until reproduci-
ble scans were observed. The electrodes were rinsed with water before self
assembly (SAM) modification.
The surface modification is illustrated in Fig. 1. An aliquot (5 ml) of probe
DNA (5.66 m mol l21) solution was pippted onto the surface of AuE. The
electrode was allowed to dry overnight for approximately 15 h (at room
28 Z. Wang et al.

Figure 1. The preparation and sandwich hybridization schematic illustration of DNA

biosensor.

temperature). Subsequently, the AuE/SAM was rinsed vigorously with PBS.

Then, the electrode was immersed in a 0.001 mol l21 thioglycolic acid
solution for 1 h to reduce the generally accessible surface area besides the
available probe DNA eptitopes.

Hybridization of DNA-modified Electrodes

The DNA-modified electrode was immersed in hybridization buffer solution

(STE, pH 8.8) containing the desired amount of target DNA for 30 min at
658C. Subsequently, the electrode was soaked in a stirred PBS for removing
nonspecifically absorbed target DNA. Then, an aliquot (5 ml) of HRP-DNA
was pippted onto the surface of the electrode and incubated for 30 min at
658C. The electrode was then thoroughly rinsed in a stirred PBS. Thus, a
hybrid modified AuE was obtained.

Measurement

Amperometric measurements were performed in an electrochemical cell

holding 10 ml of the supporting electrolyte containing 1.0 mmol ml21 hydro-
quinone. A three-electrode system was used at an applied potential of
2150 mV versus SCE. After the background current was stabilized, the
response was recorded after addition of H2O2.
Electrochemical DNA Biosensor Based on HRP-labeled Probe 29

Figure 2. Cyclic voltammograms obtained with the DNA biosensor in an unstirred

0.067 mol l21 PBS (pH 7.00) containing 1.0 mmol l21 hydroquinone with (b) and without
(a) addition of H2O2 to a final concentration of 2.0 mmol l21 (b). Scan rate 100 mV sec21.

RESULTS AND DISCUSSION

Electrochemical Characterization of the DNA Biosensor

The operation of amperometric DNA sensor was based on the use of detection
DNA labeled with enzyme HRP and H2O2 as the substrate. Hydroquinone, one
of the best electron transfer mediator for HRP (Volpe et al. 1998), is chosen as
the electron transfer mediator. In the presence of HRP, H2O2 oxidizes hydro-
quinone to benzoquinone. Figure 2 shows the cyclic voltammograms
obtained with the DNA sensor in an unstirred 0.067 mol l21 PBS (pH 7.00)
with and without addition of H2O2. One couple of oxidation/reduction peak,
which represents the cyclic voltammogram of hydroquinone, was observed in
the presence of hydroquinone (Fig. 2a). But in the presence of 2.0 mmol l21
H2O2, the cyclic voltammogram displays a dramatic enhancement of the
cathodic peak current and a concomitant decrease of the anodic peak current
(Fig. 2b). These phenomena indicate that the HRP attached to the DNA
sensor surface retained high enzymatic catalytic activity and the hydroquinone
could effectively shuttled electrons from the redox center of HRP to AuE.
Figure 3 shows results obtained with the DNA sensor prepared after incu-
bating in a hybridization solution followed by amperometric determination at
2150 mV. When the amount of target DNA varied in the hybridization
solution the response current would vary. This is the basis of amperometric
assay of target DNA.
30 Z. Wang et al.

Figure 3. Curren-time curve of an incubated DNA biosensor in 0.067 mol l21 PBS
(pH 7.00) containing 1.0 mmol l21 hydroquinone recorded at 2150 mV. After the
stabilization of background current (a– b), the solution of H2O2 is added (b) and
then the current reaches an equilibrium value (c).

Optimization of Experimental Parameters

Self-assembled Time

The amount of probe DNA attached to the AuE surface was determined by the
self-assembled time. In order to obtain a maximum response with a minimum
self-assembled time, the AuE was self-assembled with the same amount of
probe DNA but different time at room temperature. Fig. 4 shows the current
response of the DNA sensor increased up to 15 h and then tends to change
only slightly. So, the self-assembled time of 15 h was routinely employed in
these assays.

Hybridization Time

The effect of hybridization time on amperometric signals was also investi-

gated. When the targets DNA in the hybridization buffer solution reach the
probe DNA at the surface of the DNA sensor, it takes time for the contacting
species to form dsDNA. Figure 5 demonstrates with the prolonging hybridiz-
ation time from 0.25 to 2.5 h, the amperometric current increases dramatically
and then tends to change only slightly. So an incubation time of 30 min was
adopted in experiments. One would expect that most of the surface-exposed
probes DNA are binding with the target DNA in the hybridization solution,
forming compact complexes on the surface of the DNA sensor.
Electrochemical DNA Biosensor Based on HRP-labeled Probe 31

Figure 4. Effect of self-assembled time.

Hybridization Temperature

An additional parameter that affected the assay was the hybridization tempera-
ture. The effect of hybridization temperature on response current was
examined from 25 to 658C (Fig. 6). It was found that the signal increases
with an increase of temperature up to 658C. At higher temperature the
dsDNA would untie, 658C has been chosen as the optimum temperature for
the hybridization of the DNA sensor.

The pH of the Substrate Solution

The pH of the substrate solution, which has significant influence on the

reduction of the enzyme catalytic product, has been optimized (Fig. 7). It is

Figure 5. Effect of hybridization time. The DNA biosensor was incubated in STE
buffer solution containing 1165 pmol l21 target DNA at 658C.
32 Z. Wang et al.

Figure 6. Effect of hybridization temperature. The DNA biosensor was incubated 30

min in STE buffer solution containing 1165 pmol l21 target DNA.

observed that the current response increases with an increase of the pH of the
substrate solution up to pH 7.00, and then it decreases at higher pH. So, pH
7.00 of the substrate solution was chosen as the optimum for the assays.

Calibration Curve

Figure 8 shows the calibration curve obtained using target DNA standards
under optimal experimental conditions. The response signal increases
linearly with the increase of the logarithm of the target DNA concentration
in the range of 1.17
10211 21.17
1027 mol l21 with a correlation coeffi-
cient value of 0.964. The linear regression equation is I ¼ 22.54 þ 3.67 log
CDNA with a detection limit of 5.85
10212.

Figure 7. Effect of the pH of the substrate solution.

Electrochemical DNA Biosensor Based on HRP-labeled Probe 33

Figure 8. Calibration curve of the DNA biosensor. The DNA biosensor was incu-
bated in SET buffer solution of pH 7.00 containning different amount of target
DNA. Other conditions are optimal experimental conditions.

The Selectivity of the DNA Sensor

The selectivity of this assay was explored by using the AuE/SAM to

hybridize with different kinds of DNA sequences. Figure 9 shows amperometric
response of the DNA sensor to 2325 pmol l21 random oligonucleotide (a),
1600 pmol l21 four-base-mismatched oligonucleotide (b), and 1165 pmol l21
perfectly matched oligonucleotide (c). The sensor have much smaller
amperometric responses when hybridization with four-base-mismatched

Figure 9. Amperometric responses upon adding 2.0 mmol L21 H2O2 to PBS of
2325 pmol l21 random oligonucleotide (a), 1600 pmol l21 four-base-mismatched
oligonucleotide (b), and 1165 pmol l21 perfectly matched oligonucleotide (c).
34 Z. Wang et al.

oligonucleotide and random oligonucleotide. As expected, only the complemen-

tary target sequence gave a significant increasing response, which shows the
high selectivity of this hybridization assay. The high sequence-selectivity of
this sensor is double-guaranteed by the complementary hybridization of target
DNA with capture and detection probes.

The Regeneration of the DNA Sensor

Regeneration of ssDNA and removal of target DNA and detection DNA were
achieved by rinsing the electrode in hot hybridization buffer (1008C) for 5 min.
The electrode after regeneration can be used again to detect target DNA.

Reproducibility of the DNA Sensor

Reproducibility of the DNA sensor was tested by measuring 1165 pmol l21
target DNA solution. Four DNA sensors, made independently, showed the
response current values of 6.78, 6.43, 6.62 and 6.85 mA with an acceptable
variation coefficient of 2.83% (n ¼ 4).

CONCLUSIONS

In this paper, probe DNA-SH was adsorbed on an AuE surface, forming a

DNA-modified electrode. By using HRP-DNA as detection probe, a sequential
sandwich format was performed to detection gene which is associated with
colorectal cancer. The simple manipulation procedure, high selectivity, low-
cost, fast response and broad linear range are the main features of proposed
DNA sensor. Through removal of target DNA and detection DNA in hot
hybridization buffer, the sensor can be regenerated. This can be used as a
platform to develop a method to diagnose cancer in its early stage, which is
favorable for the treatment.

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