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Cite this: Chem. Commun., 2012, 48, 6390–6392
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An ultrasensitive electrochemical impedance sensor for a special BRCA1
breast cancer gene sequence based on lambda exonuclease assisted target
recycling amplificationw
Huifeng Xu,ab Lili Wang,ab Hongzhi Ye,a Lishuang Yu,a Xi Zhu,b Zhenyu Lin,b
Guangwen Wu,a Xihai Li,a Xianxiang Liu*a and Guonan Chen*b
Published on 20 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CC31588B
Downloaded by North Carolina State University on 04 December 2012
Received 2nd March 2012, Accepted 20th April 2012
DOI: 10.1039/c2cc31588b
A label-free, target recycling electrochemical impedance spectro-
scopy (EIS) DNA sensor has been developed for detection of a
model related to the BRCA1 breast cancer gene with a detection
limit of 0.05 nM.
Rapid and simple detection of ultralow concentrations of specific
DNA sequences is becoming an important sensing methodology
in medical diagnostics, pathogen detection, forensic analysis, and
environmental monitoring.1 In recent years, DNA sensors based
on optical2 or electrochemical3 detection have been developed.
Due to the fact that electrochemical detection is simple, portable,
sensitive, fast response and low cost, it has become a promising
tool to develop electrochemical DNA sensors.4 These sensors
can be prepared by immobilizing single-stranded DNA (ssDNA)
on the electrodes to recognize its complementary DNA target
sequence by hybridization.5 Electroactive indicators6 or other
methods7 are used to examine the hybridization events. Although
above electrochemical DNA sensors have been used to analyze
DNA successfully, their capacity to detect trace amounts of DNA
is still needed to improve.
Very recently, target recycling based on the DNA enzyme
has become an interesting alternative for sensitive detection of
DNA.7b,8 In this approach, target–probe hybridization triggers the
selective DNA enzymatic digestion of the signaling probe, and then
the released intact target DNA continues to initiate the digestion of
other probe molecules, thereby generating multiple signaling events
and achieving signal amplification. Among various strategies,
restriction endonucleases and exonuclease III were mainly utilized,
and sensors based on which were developed.7b,8b,e,f Despite the
great progress made by these target recycling DNA sensors, some
limits still exist. For example, the restriction endonucleases require
a
Academy of Integrative Medicine, Fujian University of Traditional
Chinese Medicine, Fuzhou, Fujian, P. R. China.
E-mail: liuxianxiang@163.com
b
Ministry of Education Key Laboratory of Analysis and Detection for
Food Safety, Fujian Provincial Key Laboratory of Analysis and
Detection Technology for Food Safety, Department of Chemistry,
Fuzhou University, Fuzhou, Fujian 350002, P. R. China.
E-mail: gnchen@fzu.edu.cn; Fax: +86-591-22866135;
Tel: +86-591-22866135
w Electronic supplementary information (ESI) available: Experimental
section. See DOI: 10.1039/c2cc31588b
6390 Chem. Commun., 2012, 48, 6390–6392
COMMUNICATION
a specific sequence of nucleotides for recognition, causing the
limitation in the detection of only target sequences containing
the recognition units.8d While exonuclease III, a well known
exonuclease, can catalyze the stepwise removal of mononucleotides
from 30 -hydroxyl termini (30 -blunt end) of double-stranded DNA
(dsDNA).8a However, the clinical samples would require the probe
to target an interior region of dsDNA (not necessarily producing a
preferred 30 -blunt end for enzymatic recognition).
Alternatively, lambda exonuclease becomes an interesting
toroidal enzyme. It can processively degrade one strand of a
dsDNA in the 5 0 to 3 0 direction, where a 5 0 -phosphate terminus
is required for enzymatic recognition and initiation.9 Only in
the presence of the co-factor Mg2+, this enzyme is selective for
double-stranded over single-stranded substrates. However, till
now DNA sensors using lambda exonuclease are still very
few.8c,10 Hsieh et al.10 proposed an electrochemical DNA sensor
based on the phenomenon of target recycling. In this sensor
methylene blue was situated at the center of capture DNA 1
(molecular beacon, as the substrate), which would increase the
cost of the sensor and complicate the procedure.
Herein, we demonstrate a simple, label-free and more
versatile target recycling DNA sensor using lambda exonuclease.
In this study the BRCA1 breast cancer gene is chosen as a model
target, whose mutations may induce breast cancer (7% of breast
cancer cases come from the mutation of BRCA1 gene).11
Different from the above alternating-current voltammetry,10
electrochemical impedance spectroscopy (EIS) is used as a
detection technique. Because of its ability for probing the
interfacial property at the electrode surface and the possibility
of performing label-free detections, EIS is increasingly used for
detection of biorecognition events at the electrode surface.12
Additionally, compared with the previous one,10 this can save
the cost greatly without a reagent-labeled substrate. Based on this
strategy, BRCA1 breast cancer gene amplified detection could be
achieved with ultrahigh sensitivity and good specificity.
The principle of a target recycling impedimetric DNA
sensor based on lambda exonuclease is described in Fig. 1.
Capture DNA 1 contains 19-nucleotide (nt) that is modified
with a thiol group at its 3 0 terminus and a phosphate group at
its 5 0 terminus. It is immobilized on the gold electrode through
thiol–Au interaction. In the absence of target DNA, capture
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and 0.2 units mL1 exonuclease mixture buffer, capture DNA 1

immobilized on the electrode is digested by exonuclease, and then
the negative charge on the electrode decreases sharply, thus, the
reaction of Fe(CN)63/4 on the electrode is convenient, bringing
on a significantly decreased Rct value (curve d, 882 O). In the
control experiment, capture DNA 2 without PHOS is immobilized
on the electrode, and hybridized with target DNA. The presence
of exonuclease could not decrease the Rct (see Fig. S2, ESIw).
Above results indicate that, firstly, the capture DNA 1 can be
successfully immobilized on the electrode and hybridized with
target DNA, and secondly, lambda exonuclease only affects the
dsDNA with a 50 phosphate; consequently, it is feasible to use
lambda exonuclease to assay target DNA based on EIS.
Published on 20 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CC31588B

Fig. 1 Principle of a target recycling DNA sensor. To further demonstrate the reliability of EIS measurement,
CVs of Fe(CN)63/4 on different modified electrodes are
Downloaded by North Carolina State University on 04 December 2012

DNA 1 on the electrode forms a compact negatively charged

studied. As can be seen from Fig. 2B, stepwise modification
layer due to its phosphate backbones, which prevents the
on the gold electrode is accompanied by the changes in the
negatively charged redox probe Fe(CN)63/4 from reaching
amperometric response, as well as the separation between the
the gold electrode,13 resulting in a large charge-transfer resistance
cathodic and anodic peak of the redox probe Fe(CN)63/4.
(a large semicircular). While in the presence of target DNA and
For the bare electrode, a well-defined redox pair of redox
lambda exonuclease, firstly target DNA hybridizes with capture
peaks was observed (curve a in Fig. 2B), showing the excellent
DNA 1 to form dsDNA. Subsequently, the dsDNA is selectively
electron-transfer kinetics. After capture DNA 1 is immobilized
hydrolyzed by lambda exonuclease from 50 phosphate in the
on the electrode (Fig. 2B, curve b), the amperometric response
50 -to-30 direction. After the digestion, intact target DNA is
decreases and the peak-to-peak separation enlarges. Only in the
released, which is able to hybridize with another capture DNA 1
presence of target DNA, a similar CV is observed (Fig. 2B, curve c),
and catalyze a new cycle. Finally, all capture DNA 1 on the
showing that the exonuclease does not digest capture DNA 1.
electrode is digested, thus, the negatively charged layer is destroyed,
After the interaction between target DNA and exonuclease, the
resulting in a small charge-transfer resistance (a small semicircular).
peak current significantly increases (Fig. 2B, curve d) because of
Thus, the resistance decreases with the increasing of target DNA.
the decrease of negative charge on the electrode. These CV
Hence, the concentration of target DNA can be obtained indirectly.
changes are consistent with above EIS changes, which further
To assay the feasibility of this proposed sensor, the properties
confirm the conclusion from EIS experiment.
of different modified electrodes are characterized by EIS, as
Under the optimum conditions (see ESIw for detail), the
shown in Fig. 2A. The Rct value is obtained by the equivalent
relationship between the Rct value and the concentrations of
circuit (the inset of Fig. 2, where every symbol is shown as above).
target DNA is studied. Fig. 3(A) shows the Nyquist plots
For the bare gold electrode (curve a, 45.7 O), the EIS exhibits a
dependent on different concentrations of target DNA. With
very small semicircular domain, indicating a fast charge-transfer
the increasing target DNA, more dsDNA forms, and then is
process at such an electrode. When capture DNA 1/MCH is
digested by exonuclease, resulting in the reduction of the
immobilized on the electrode, the Rct value of the electrode
negative charge on the electrode and decreasing semicircles
obviously increases (curve b, 3854 O) because the negatively
(decreasing Rct value). It is found that DRct has a relationship
charged phosphate backbone on the electrode prevents the redox
with concentrations of target DNA in the range of 0.1–10 nM
of Fe(CN)63/4 on the electrode. In the absence of target DNA,
(inset of Fig. 3). The regression equation is
lambda exonuclease is incapable of digesting the capture DNA 1,
hence, its Rct value shows a negligible change (curve c, 3882 O). Y = 13223.57 + 1273.47X R = 0.9943
After interaction with the buffer containing 1 nM target DNA
where Y is the change of Rct (DRct), X is the logarithm of target
DNA concentration, R is the regression coefficient. The detection

Fig. 2 (A) Nyquist plots and (B) cyclic voltammetries of different

electrodes. (a) Bare gold electrode; (b) capture DNA 1/MCH modified Fig. 3 (A) Electrochemical impedance spectroscopy of the modified
gold electrode; (c) capture DNA 1/MCH modified gold electrode after electrode after interaction of different concentrations of target DNA.
interaction with lambda exonuclease; (d) dsDNA/MCH modified gold (B) The linear relationship between impedance and logarithmic target
electrode after interaction with lambda exonuclease. concentration.

This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 6390–6392 6391

limit of this method is 0.042 nM (defined as S/N = 3), which
is superior to those of other methods based on lambda
exonuclease,8c,10 and other DNA sensors for the detection of
BRCA1 breast cancer gene.14 Compared with the detection
limit of the sensor without exonuclease (0.2 nM, data not
shown), about 4.5 times improvement is obtained, which
indicates that the presence of lambda exonuclease leads to
target recycling amplification.
Above data were obtained at least 5 times. It is found that
the relative standard deviations (RSD) of Rct are in the range
of 1.2% to 1.8%. Moreover, after the test, the electrodes are
polished and remodified. Under the same conditions, 6.2% of
Rct change can be achieved compared with the previous
Published on 20 April 2012 on http://pubs.rsc.org | doi:10.1039/C2CC31588B
measurement. These results show that this target recycling
Downloaded by North Carolina State University on 04 December 2012
DNA sensor has good stability and regeneration.
The selectivity of the present biosensor was investigated by
using capture DNA 1 to hybridize with the same concentration
(1 nM) of the complete complementary target DNA sequence
(histogram a), the single-base mismatched DNA sequence
(histogram b) and the noncomplementary DNA sequence (histo-
gram c), respectively. The result is shown in Fig. S4 (ESIw).
The response to noncomplementary DNA is lowest, while the
response to single base mismatched DNA is 66.7% lower than
that of target DNA. The result shows that this sensor has
good hybridization specificity compared to other DNA sensors.
However, there is a little interference from single base mismatched
DNA when the single base mismatch sets in the middle
of mismatch DNA, for only half of capture DNA on the
electrode can be digested after the hybridization, which results
in a quite DRct.
In summary, a novel exonuclease-induced target recycling
DNA sensor based on EIS is developed. In the presence of
lambda exonuclease, hybridization of capture DNA 1 on the
electrode with the target DNA (BRCA1 breast cancer gene)
results in a larger |DRct| than that of without exonuclease.
Moreover, this sensor can easily discriminate the complementarity
between single-base mismatched DNA and non-complementary
DNA. Hence, this sensor is promising to be applied for early
diagnosis of BRCA1 breast cancer. Additionally, it is envisioned
that this strategy will have a promising future for investigation of
DNA hybridization and will play the potential predominance in
diagnosis of virus or disease.
This project was financially supported by the National Basic
Research Program of China (No. 2010CB732403), the NSFC
(41076059), the Key Foundation of Education Department of
Fujian Province (JK2009016), the Natural Sciences Foundation
of Fujian Province (2011J01035), and the Program for
Changjiang Scholars and Innovative Research Team in University
(No. IRT1116).
6392 Chem. Commun., 2012, 48, 6390–6392
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Notes and references
1 A. Sassolas, B. D. Leca-Bouvier and L. J. Blum, Chem. Rev., 2008,
108, 109.
2 (a) D. R. Walt, Science, 2000, 287, 451; (b) N. Kumar, A. Dorfman
and J. Hahm, Nanotechnology, 2006, 17, 2875; (c) H. A. Ho,
M. Béra-Abérem and M. Leclerc, Chem.–Eur. J., 2005, 11, 1718.
3 (a) J. Wang, G. Liu and A. Merkocçi, J. Am. Chem. Soc., 2003,
125, 3214–3215; (b) M. T. Castaneda, A. Merkocçi, M. Pumera
and S. Alegret, Biosens. Bioelectron., 2007, 22, 1961.
4 A. Sassolas, B. D. Leca-Bouvier and L. J. Blum, Chem. Rev., 2008,
108, 109.
5 (a) T. G. Drummond, M. G. Hill and J. K. Barton, Nat. Biotechnol.,
2003, 21, 1192; (b) J. Wang, G. Rivas, X. Cai, E. Palecek, P. Nielsen,
H. Shiraishi, N. Dontha, D. Luo, C. Parrado and M. Chicharro,
Anal. Chim. Acta, 1997, 347, 1; (c) C. Fan, K. W. Plaxco and
A. J. Heeger, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 9134;
(d) J. Zhang, S. Song, L. Wang, D. Pan and C. Fan, Nat. Protocols,
2007, 2, 2888; (e) H. F. Teh, H. Q. Gong, X. D. Dong, X. T. Zeng,
A. L. K. Tan, X. H. Yang and S. N. Tan, Anal. Chim. Acta, 2005,
551, 23; (f) F. Le Floch, H. A. Ho, P. Harding-Lepage, M. Bedard,
R. Neagu-Plesu and M. Leclerc, Adv. Mater., 2005, 17, 1251.
6 (a) K. M. Millan and S. R. Mikkelsen, Anal. Chem., 1993, 65, 2317;
(b) J. Wang, X. Cai, G. Rivas, H. Shiraishi, P. A. M. Farias and
N. Dontha, Anal. Chem., 1996, 68, 2629; (c) N. Zhu, A. Zhang,
Q. Wang, P. He and Y. Fang, Anal. Chim. Acta, 2004, 510, 163;
(d) X. M. Li, H. Q. Ju, C. F. Ding and S. S. Zhang, Anal. Chim.
Acta, 2007, 582, 158.
7 (a) J. Wang, G. Rivas, J. R. Fernandes, J. L. Lopez Paz, M. Jiang and
R. Waymire, Anal. Chim. Acta, 1998, 375, 197; (b) H. Peng, C. Soeller,
N. Vigar, P. A. Kilmartin, M. B. Cannell, G. A. Bowmaker,
R. P. Cooney and J. Travas-Sejdic, Biosens. Bioelectron., 2005, 20, 1821.
8 (a) J. Chen, J. Zhang, J. Li, F. Fu, H. H. Yang and G. Chen, Chem.
Commun., 2010, 46, 5939; (b) H. J. Lee, Y. Li, A. W. Wark and
M. Robert, Anal. Chem., 2005, 77, 5096; (c) X. Zuo, F. Xia,
Y. Xiao and K. W. Plaxco, J. Am. Chem. Soc., 2010, 132, 1816;
(d) R. Fu, T. Li, S. S. Lee and H. G. Park, Anal. Chem., 2011,
83, 494; (e) J. A. Dougan, D. MacRae, D. Graham and K. Faulds,
Chem. Commun., 2011, 47, 4649; (f) T. Kiesling, K. Cox,
E. A. Davidson, K. Dretchen, G. Grater, S. Hibbard,
R. S. Lasken, J. Leshin, E. Skowronski and M. Danielsen, Nucleic
Acids Res., 2007, 35, e117; (g) E. Mokany, S. M. Bone,
P. E. Young, T. B. Doan and A. V. Todd, J. Am. Chem. Soc.,
2009, 132, 1051.
9 (a) P. G. Mitsis and J. G. Kwagh, Nucleic Acids Res., 1999,
27, 3057; (b) J. W. Little, Gene Amplif. Anal., 1981, 2, 135.
10 K. Hsieh, Y. Xiao and H. Tom Soh, Langmuir, 2010, 26, 10392.
11 (a) J. D. Fackenthal and O. I. Olopade, Nat. Rev. Cancer, 2007,
7, 937; (b) E. Honrado, A. Osorio, J. Palacios and J. Benitez,
Oncogene, 2006, 25, 5837; (c) S. Armaou, I. Konstantopoulou,
T. Anagnostopoulos, E. Razis, I. Boukovinas, N. Xenidis,
G. Fountzilas and D. Yannoukakos, Eur. J. Cancer, 2007, 43,
443–453.
12 (a) B. Pejcic and R. D. Marco, Electrochim. Acta, 2006, 51, 6217;
(b) I. Willner and M. Zayats, Angew. Chem., Int. Ed., 2007,
46, 6408.
13 M. Cho, S. Lee, S. Y. Han, J. Y. Park, M. A. Rahmen, Y. B. Shim
and C. Ban, Nucleic Acids Res., 2006, 34, e75.
14 (a) J. Wang, D. Xu, A. Erdem, A. Merkoci, R. Polsky and
M. A. Salazar, Talanta, 2002, 56, 931; (b) M. T. Castañeda,
A. Merkoçi, M. Pumera and S. Alegret, Biosens. Bioelectron.,
2007, 22, 1961.
This journal is c The Royal Society of Chemistry 2012