SCIENCE CHINA
Chemistry
• ARTICLES •
March 2015
Vol.58 No.3: 514–518
doi: 10.1007/s11426-014-5258-9
A highly sensitive chemiluminescence sensor for detecting mercury (II)
ions: a combination of Exonuclease III-aided signal amplification
and graphene oxide-assisted background reduction
Yang Tian1, Yue Wang1, Yan Xu3, Yang Liu2, Di Li3* & Chunhai Fan3*
1
2
College of Life and Environmental Science, Minzu University of China, Beijing 100081, China
Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China
3
Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics,
Chinese Academy of Sciences, Shanghai 201800, China
Received June 30, 2014; accepted August 7, 2014; published online January 6, 2015
In this paper, we report a highly sensitive chemiluminescence (CL) sensor for Hg2+ ions based on thymine-Hg2+-thymine
(T−Hg2+−T) coordination chemistry. We designed a thymine rich oligonucleotide as a capture probe and a signal probe that includes two functional domains: a horseradish peroxidase-mimicking DNAzyme domain for the generation of CL, and a recognition domain. Graphene oxide (GO) was introduced to adsorb the signal probe via - interaction, which brought the
DNAzyme domain and GO into close proximity and quenches CL. In the presence of Hg2+ ions, the coordination of Hg2+ with
the capture probe yielded a hairpin complex, triggers cascaded strand displacement reactions and Exonuclease III-assisted signal amplifications. As a result, accumulated amounts of DNAzyme were generated and released from GO, leading to an enhanced CL signal. This strategy combines enzyme-based signal amplification and GO as a background reducer, leads to a limit
of detection (LOD) of 2 nmol/L. This simple detection system provides a label-free yet sensitive approach for detection of
Hg2+ ions.
graphene oxide, chemiluminescence, mercury (II) ions, DNAzyme
1 Introduction
Mercury ions, the most stable form of inorganic mercury,
have been widely recognized as highly toxic environmental
pollutants. It has also been proved that serious medical effects are caused due to its accumulative properties in the
environment [1,2]. Thus, monitoring the levels of Hg2+ ions
in aquatic ecosystems is highly important. There have been
numerous reports on electrochemical Hg2+ sensors by using
anodic stripping voltammetry [3–5]. However, most of
these electrochemical sensors could hardly provide a limit
of detection (LOD) as low as 10 nmol/L (2 ppb), a toxic
*Corresponding authors (email: [email protected]; [email protected])
© Science China Press and Springer-Verlag Berlin Heidelberg 2015
level of Hg2+ designated by the U.S. Environmental Protection Agency (EPA).
Recently, the coordinative interaction between Hg2+ and
bis-thymine, as demonstrated by Ono and coworkers [6,7],
has stimulated new approaches in creating DNA-based Hg2+
sensors. Many fluorescent [8–10], colorimetrical [11–14]
and electrochemical sensors [15,16] with excellent performances have been developed. To improve the sensitivity of
the thymine-based Hg2+ sensor, several signal amplification
strategies including strand displacement amplification [11]
and cascade enzymatic signal [17–19], have been introduced. These strategies, however, require a specific sequence for the enzyme to recognize. To overcome this
shortcoming, Exonuclease III (Exo III), a nuclease that catalyzes the stepwise removal of mononucleotides from
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Tian et al.
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3′-hydroxyl termini of duplex DNAs, was introduced [20].
Accordingly, a sequence-independent strategy, Exo IIIaided signal amplification, was developed to “recycle” target molecules, resulting in improved sensitivity [21–23].
The Exo III-aided signal amplification was first developed for the fluorescent DNA sensor by using a molecular
beacon as the readout [20]. Herein, we propose a modified
version of the Exo III-aided signal amplification to develop
a chemiluminescent assay for Hg2+ ions. Unlike previously
reported fluorescent assay, we introduce a horseradish
peroxidase-mimicking DNAzyme to generate chemiluminescence (CL) in order to further improve the sensitivity
[24,25]. In addition, we introduce graphene oxide (GO)
[26,27] as a background reducer by employing two intrinsic
properties of GO: (1) the different affinity of GO towards
single stranded (ss-) and double stranded (ds-) DNA [28,29];
(2) the strong quenching of CL by GO [30–32]. Our results
indicated that the proposed CL sensor provides both high
selectivity and sensitivity for Hg2+ ions, which could lead to
the development of a signal amplification platform for label- free detection of functional DNA-based analytes.
2 Experimental
2.1
Materials and reagents
Hg(ClO4)2 was purchased from Aldrich (USA) and used as
received. Exo III was purchased from New England Biolabs
(UK). GO was synthesized from natural graphite powder by
a modified Hummers method. Other chemicals were of analytical purity and used as received.
Oligonucleotides were synthesized by Sangon Biotech
(China) and purified with HPLC. The sequences of oligonucleotides used in this work are as follows:
Capture probe (1): 5′-TTTCTTGTTTGTTTGTTGGCCCCCCTTCTTTCTTACTTGTTA-3′
Helper strand (2): 5′-TCCAGTAAGAAAGAAGGGGCAT-3′
Signal probe (3): 5′-GGGTAGGGCGGGTTGGGATAC*CCCCTTCTTTCT-3′ (* is a phosphorothioated nucleotide).
2.2 Procedures for Hg2+ detection
The capture probe 1 and helper strand DNA 2 were first
hybridized to form a duplex. Briefly, equal amount of 1 and
2 (0.5 mol/L, each) were mixed in a Tris-Ac buffer solution (20 mmol/L) containing NaAc (200 mmol/L, pH 8.0).
The mixture was then heated to 95 °C for 5 min and allowed
to cool to room temperature. Next, Hg2+ of different concentrations were incubated with 1/2 duplex at room temperature for another 1 h (Solution A). All reactions were processed in a PCR tube (0.2 mL) and the total volume of the
reaction was 0.1 mL.
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GO (1 mg/mL, 10 L) and the signal probe 3 (0.5
mol/L) were incubated at room temperature for 1 h (Solution B) in another PCR tube. Then Solution A and B were
mixed and K+ ions (10 mmol/L) were added to the resulting
mixture to facilitate the formation of G-quadruplex. The
addition of Exo III (2 U) to the above mixture, followed by
incubating at 37 °C for 1 h initiated the Exo III-aided signal
amplification. Finally, hemin (1 mol/L) was added to the
resulting mixture to facilitate the formation of DNAzyme.
2.3
CL measurements
The CL measurements were performed by adding luminol
(1 mmol/L) and H2O2 (2 mmol/L) to the resulting mixture.
The sample solution was then injected into the carrier
stream (water), through which the mixture of sample, luminol and H2O2 solution finally reached the detector to produce CL signals.
2.4
Apparatus
The CL measurement was carried out on an EnVision Multilabel Plate Readers (PerkinElmer, USA).
3
Results and discussion
3.1 Principle of the proposed Hg2+ sensor
The detailed principle of the proposed Hg2+ sensor is outlined in Figure 1. We designed a T-rich oligonucleotide 1 as
capture probes for Hg2+. The capture probe was first hybridized with the helper strand 2 to form a duplex. We also
designed a signal probe 3. The signal probe 3 included two
regions. Region I (blue) was a G-quadruplex domain, which
formed a horseradish peroxidase-mimicking DNAzyme
upon stacking by Hemin. Region II (red) included the sequence that is complementary to part of 2. A point of note is
that we did not include K+ in the incubation buffer, thus
region I could not form G-quadruplex. Therefore, 3 still
remained as a flexible random coil and was attached to GO
via - interaction. Upon the addition of Hg2+, the complex
of Hg2+ with thymine sites in 1 yielded a hairpin complex,
which resulted in the release of 2 from the duplex. The released 2 could be further hybridized with cognition domain
II in 3.
Since GO possesses weaker interaction with ds-DNA, the
2/3 duplex was then liberated from GO and resulted in a
recessed 3′ terminus in 3. Then K+ was introduced into the
sample solution to facilitate the formation of G-quadruplex.
Exo III catalyzed the stepwise removal of mononucleotides
from 3′ hydroxyl termini of the duplex DNAs and its preferred substrates were blunt or recessed 3′-termini. Particularly, Exo III was not active on phosphorothioate-linked
nucleotides. In the present work, we rationally encoded a
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Figure 1
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(color online) The detailed detection scheme of the proposed Hg2+ sensor.
phosphorothioated cytosine in 3 (in black letter). It proves
that Exo III catalyzes the stepwise removal of mononucleotides in 3 from recessed 3′ terminus until the phosphorothioated cytosine, liberats G-quadruplex in 3 and releases 2
from the 2/3 duplex. The released 2 could then hybridize
with a second 3, which is attached to GO, initiating the Exo
III-aided signal amplification, leading to the generation of
accumulated G-quadruplex. In addition, Exo III also reveals
some weak catalytic activities to stepwise digest the Hg2+
mediated hairpin complex (1). As a result, more Hg2+ ions
are from the T–Hg2+–T base pair during the digestion process, which further folding a new undigested hairpin probe,
thus starting a new cycle of digestion. Therefore, the synthetic effect of Exo III leads to recycling signal amplification that produces accumulated amounts of G-quadruplex.
The produced G-quadruplex, upon stacking by hemin,
forms DNAzyme that catalyze the CL reaction between
luminol and H2O2.
3.2
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3/GO mixture, the hybridization of 2 and 3 extracted the
G-quadruplex from GO surface. As a result, CL emission
recovered to 80% of 3 (Figure 2(c)). The decrease and recovery of CL emission indicate that GO is an effective
CL-quencher. And similar to its quenching of fluorescence,
the quenching mechanism of CL by GO is partly contributed to by either electron transfer or resonance energy transfer.
Hence, the quenching is distance-dependent, i.e. GO
quenches the CL generated by the surface attached DNAzyme. Once DNAzyme is released from the GO surfaces, the
CL is recovered accordingly. In other words, GO functions
Investigation of the quench of CL by GO
It should be noted that K+ ions are present in the buffer solution during the Exo III-aided signal amplification. Hence,
region I of 3 that is attached to GO, still forms G-quadruplex DNAzyme and generates CL signal as background. We
thereby first investigated the quenching of CL by GO. Upon
incubation with GO, the CL of 3 dropped dramatically
(Figure 2(a, b)). Interestingly, upon the addition of 2 to the
Figure 2 Chemiluminescence profiles of luminol generated by 3 (a),
3/GO mixture (b) and 3/GO mixture+2 (c). Experimental conditions: 2, 500
nmol/L; GO, 10 g; 3, 500 nmol/L; luminol, 1 mmol/L; H2O2, 2 mmol/L.
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as an effective background reducer and only the Hg2+liberated DNAzyme could generate effective CL readout.
3.3 Interrogation of the efficiency of Exo III-aided
signal amplification
We tested the efficiency of Exo III-aided signal amplification upon interrogation with 0 and 40 nmol/L of Hg2+, respectively. Figure 3 shows the CL profile of the system upon response to Hg2+ (40 nmol/L) without (curve (c)) and
with (curve (d)) the aid of Exo III, while curves (a) and (b)
are the background noise of the system without and with the
aid of Exo III, respectively. Clearly, the introduction of Exo
III significantly enhanced the CL signal, suggesting that
more DNAzymes were generated by the Exo III-induced
recycling. In addition, with the aid of Exo III signal amplification, background noise of the system only underwent
negligible increase (curves (a) to (b)), which further conforms the strong quenching ability of GO to suppress noise
during signal amplification cycles.
3.4
The sensing performance of the proposed strategy
With the knowledge that the proposed strategy possesses a
synthetic effect of signal amplification and background reduction, we challenged this system with Hg2+ to test its
sensing performance. Figure 4 depicts the CL signals upon
challenging the system with different concentrations of Hg2+.
As the concentrations of Hg2+ increased, the intensity of the
resulting CL responses increased. The resulting calibration
curve for Hg2+ is shown in the inset of Figure 4, and the
LOD was calculated as 2 nmol/L (0.4 ppb) (>3), which
satisfactorily meets the sensitivity requirement of EPA.
3.5 Examination of the selectivity of the proposed sensor for detection of Hg2+
We further demonstrated that this assay is selective toward
Figure 3 Chemiluminescence profiles of the system upon response to
Hg2+ (40 nmol/L) without (c) and with (d) the aid of Exo III, while (a) and
(b) are the background noise of the system without and with the aid of Exo
III.
Figure 4 Chemiluminescence profiles of luminol generated by the sensing system on being challenged with different concentrations of Hg2+. (a–g)
0, 5, 10, 20, 40, 60 and 100 nmol/L, respectively.
Hg2+. We systemically challenged the assay with 8 interference metal ions. Figure 5 compares the CL signals of interference metal ions at high concentrations (1 mol/L) and
Hg2+ (100 nmol/L). Clearly, Hg2+ ions could be easily differentiated despite the presence of all other metal ions present with a 10-fold higher concentration.
4 Conclusions
We employed an Exo III-aided signal amplification system
for the detection of Hg2+. Differing from a molecular beacon, we designed a signal probe containing DNAzyme that
catalyzes the reaction between luminol and H2O2, which
generates CL signal as the readout. The introduction of GO
enables the efficient suppression of background noise during signal amplification owing to its quenching ability and
higher affinity to ss-DNA. The proposed assay reveals synthetic advantages of multiple signal amplification (Exo
III-aided signal recycling and DNAzyme-based catalytic
Figure 5 Selectivity of the analysis of Hg2+ ions by the method depicted
in Figure 1. The concentration of Hg2+ was 100 nmol/L and all other interference metal ions were 1 mol/L.
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chemiluminescence) and background suppression, which
enables the selective detection of Hg2+ down to 2 nmol/L.
The proposed method avoids the dual label requirement of
molecular beacon, thus significantly lowering the cost and
improving the sensitivity. Therefore, we expect it to be a
general platform for functional DNA-based sensing.
This work was supported by the National Natural Science Foundation of
China (21222508, 21375073), the Shanghai Municipal Commission for
Science and Technology (13QH1402300), the State Ethnic Affairs Commission (10ZY02) and the 111 Project of Minzu University (B08044).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Babel S, Kurniawan TA. Low-cost adsorbents for heavy metals uptake from contaminated water: a review. J Hazard Mater, 2003, 97:
219–243
Boening DW. Ecological effects, transport, and fate of mercury: a
general review. Chemosphere, 2000, 40: 1335–1351
Gao C, Huang XJ. Voltammetric determination of mercury(II).
Trac-Trend Anal Chem, 2013, 51: 1–12
Wang SP, Forzani ES, Tao NJ. Detection of heavy metal ions in water by high-resolution surface plasmon resonance spectroscopy combined with anodic stripping voltammetry. Anal Chem, 2007, 79:
4427–4432
Xu XX, Duan GT, Li Y, Liu GQ, Wang JJ, Zhang HW, Dai ZF, Cai
WP. Fabrication of gold nanoparticles by laser ablation in liquid and
their application for simultaneous electrochemical detection of Cd2+,
Pb2+, Cu2+, Hg2+. ACS Appl Mater Interfaces, 2014, 6: 65–71
Ono A, Togashi H. Highly selective oligonucleotide-based sensor for
mercury(II) in aqueous solutions. Angew Chem Int Ed, 2004, 43:
4300–4302
Miyake Y, Togashi H, Tashiro M, Yamaguchi H, Oda S, Kudo M,
Tanaka Y, Kondo Y, Sawa R, Fujimoto T, Machinami T, Ono A.
Mercury(II)-mediated formation of thymine-Hg-II-thymine base pairs
in DNA duplexes. J Am Chem Soc, 2006, 128: 2172–2173
Freeman R, Finder T, Willner I. Multiplexed analysis of Hg2+ and
Ag+ ions by nucleic acid functionalized CdSe/ZnS quantum dots and
their use for logic gate operations. Angew Chem Int Ed, 2009, 48:
7818–7821
Ye BC, Yin BC. Highly sensitive detection of mercury(II) ions by
fluorescence polarization enhanced by gold nanoparticles. Angew
Chem Int Ed, 2008, 47: 8386–8389
Zheng DM, Zou RX, Lou XH. Label-free fluorescent detection of
ions, proteins, and small molecules using sructure-switching sptamers,
SYBR gold, and exonuclease I. Anal Chem, 2012, 84: 3554–3560
Li D, Wieckowska A, Willner I. Optical analysis of Hg2+ ions by oligonucleotide-gold-nanoparticle hybrids and DNA-based machines.
Angew Chem Int Ed, 2008, 47: 3927–3931
Lee JS, Han MS, Mirkin CA. Colorimetric detection of mercuric ion
(Hg2+) in aqueous media using DNA-functionalized gold nanoparticles. Angew Chem Int Ed, 2007, 46: 4093–4096
Wang ZD, Lee JH, Lu Y. Highly sensitive “turn-on” fluorescent sensor for Hg2+ in aqueous solution based on structure-switching DNA.
Chem Commun, 2008: 6005–6007
Li T, Li BL, Wang EK, Dong SJ. G-quadruplex-based DNAzyme for
sensitive mercury detection with the naked eye. Chem Commun, 2009:
3551–3553
Zhu ZQ, Su YY, Li J, Li D, Zhang J, Song SP, Zhao Y, Li GX, Fan
CH. Highly sensitive electrochemical sensor for mercury(II) ions by
March (2015) Vol.58 No.3
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
using a mercury-specific oligonucleotide probe and gold nanoparticlebased amplification. Anal Chem, 2009, 81: 7660–7666
Xuan F, Luo XT, Hsing IM. Conformation-dependent exonuclease III
activity mediated by metal ions reshuffling on thymine-rich DNA
duplexes for an ultrasensitive electrochemical method for Hg2+ detection. Anal Chem, 2013, 85: 4586–4593
Wang F, Orbach R, Willner I. Detection of metal ions (Cu2+, Hg2+)
and cocaine by using ligation DNAzyme machinery. Chem Eur J,
2012, 18: 16030–16036
Tang LH, Liu Y, Ali MM, Kang DK, Zhao WA, Li JH. Colorimetric
and ultrasensitive bioassay based on a dual-amplification system using aptamer and DNAzyme. Anal Chem, 2012, 84: 4711–4717
Qi L, Zhao YX, Yuan H, Bai K, Zhao Y, Chen F, Dong YH, Wu YY.
Amplified fluorescence detection of mercury(II) ions (Hg2+) using
target-induced DNAzyme cascade with catalytic and molecular beacons. Analyst, 2012, 137: 2799–2805
Zuo XL, Xia F, Xiao Y, Plaxco KW. Sensitive and selective amplified fluorescence DNA detection based on exonuclease III-aided target recycling. J Am Chem Soc, 2010, 132: 1816–1818
Zuo XL, Xia F, Patterson A, Soh HT, Xiao Y, Plaxco KW. Two-step,
PCR-free telomerase detection by using exonuclease III-aided target
recycling. ChemBiochem, 2011, 12: 2745–2747
Cai S, Lao KM, Lau CW, Lu JZ. “Turn-on” chemiluminescence sensor for the highly selective and ultrasensitive detection of Hg2+ ions
based on interstrand cooperative coordination and catalytic formation
of gold nanoparticles. Anal Chem, 2011, 83: 9702–9708
Gao Y, Li BX. G-quadruplex DNAzyme-based chemiluminescence
biosensing strategy for ultrasensitive DNA detection: combination of
exonuclease III-assisted signal amplification and carbon nanotubesassisted background reducing. Anal Chem, 2013, 85: 11494–11500
Freeman R, Liu XQ, Willner I. Chemiluminescent and chemiluminescence resonance energy transfer (CRET) detection of DNA, metal
ions, and aptamer-substrate complexes using Hemin/G-quadruplexes
and CdSe/ZnS quantum dots. J Am Chem Soc, 2011, 133: 11597–11604
Liu YF, Luo M, Xiang X, Chen CH, Ji XH, Chen L, He ZK. A graphene oxide and exonuclease-aided amplification immuno-sensor for
antigen detection. Chem Commun, 2014, 50: 2679–2681
Chen D, Feng HB, Li JH. Graphene oxide: preparation, functionalization, and electrochemical applications. Chem Rev, 2012, 112:
6027–6053
Chen D, Tang LH, Li JH. Graphene-based materials in electrochemistry. Chem Soc Rev, 2010, 39: 3157–3180
He SJ, Song B, Li D, Zhu CF, Qi WP, Wen YQ, Wang LH, Song SP,
Fang HP, Fan CH. A graphene nanoprobe for rapid, sensitive, and
multicolor fluorescent DNA analysis. Adv Func Mater, 2010, 20:
453–459
Li F, Pei H, Wang LH, Lu JX, Gao JM, Jiang BW, Zhao XC, Fan CH.
Nanomaterial-based fluorescent DNA analysis: a comparative study
of the quenching effects of graphene oxide, carbon nanotubes, and
gold nanoparticles. Adv Func Mater, 2013, 23: 4140–4148
Luo M, Chen X, Zhou GH, Xiang X, Chen L, Ji XH, He ZK. Chemiluminescence biosensors for DNA detection using graphene oxide
and a horseradish peroxidase-mimicking DNAzyme. Chem Commun,
2012, 48: 1126–1128
He Y, Huang GM, Cui H. Quenching the chemiluminescence of acridinium ester by graphene oxide for label-free and homogeneous DNA
detection. ACS Appl Mater Interfaces, 2013, 5: 11336–11340
Xu SJ, Liu Y, Wang TH, Li JH. Positive potential operation of a cathodic electrogenerated chemiluminescence immunosensor based on
luminol and graphene for cancer biomarker detection. Anal Chem,
2011, 83: 3817–3823