Regulative peroxidase activity of DNA-linked hemin by graphene oxide for fluorescence DNA sensing

Article abstract of DOI:10.1039/c4cc02273d

The inhibition effect of graphene oxide toward the peroxidase activity of DNA-linked hemin was identified and conveniently utilized in the design of a homogenous fluorescence strategy for DNA sensing with high sensitivity. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc02273d

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Regulative peroxidase activity of DNA-linked
hemin by graphene oxide for fluorescence DNA
sensing†
Cite this: Chem. Commun., 2014,
50, 6714
Received 27th March 2014,
Accepted 5th May 2014
Quanbo Wang, Nan Xu, Jianping Lei and Huangxian Ju*
DOI: 10.1039/c4cc02273d
www.rsc.org/chemcomm
The inhibition effect of graphene oxide toward the peroxidase activity product in the presence of hydrogen peroxide (H2O2),7 which
of DNA-linked hemin was identified and conveniently utilized in the thus provides the possibility of amplifying the detection signal
design of a homogenous fluorescence strategy for DNA sensing in a ‘‘one target to multiple signal molecules’’ manner. This
with high sensitivity.
work designed a hemin labeled DNA probe (Table S1, ESI†) and
demonstrated the regulative peroxidase activity of DNA-linked
As an active cofactor for a variety of natural enzymes such as hemin by GO. The tunable catalysis activity could be conveni-
horseradish peroxidase and catalase, Fe(III)-protoporphyrin IX ently utilized to develop a simple homogenous fluorescence
(hemin) has attracted extensive research interest for mimicking strategy for a highly sensitive detection of DNA.
catalysis in numerous applications.1 In particular, the regulation of
The hemin labeled DNA probe was synthesized via an amide
its catalytic activity is very important for obtaining tunable detection reaction in aqueous solution, and was characterized using mass
signals and achieving high analytical sensitivity. For example, after spectroscopy (Fig. S1, ESI†) and high-performance liquid chroma-
hemin binds with the guanine quadruplex to form a DNAzyme, the tography (Fig. S2, ESI†). As shown in Scheme 1A, the probe could
peroxidase activity of hemin can be greatly enhanced compared be absorbed on the GO surface due to its flexible structure and the
with that of free hemin.2 The DNAzyme is frequently used as a p–p interaction between GO and the exposed nucleotide bases of
signal amplifying label to develop optical or electronic biosensors ssDNA, which brought hemin to the GO surface and inhibited
for DNA/RNA, proteins, and metal ions.3 To more efficiently utilize its catalytic property. The observed inhibiting effect of GO was
the catalytic function of hemin, there is an urgent need to seek an
effective way to reversibly regulate the catalytic activity of hemin.
Recently, graphene has been identified as a useful carrier to
load hemin for enhancing its catalytic properties.4 The hemin–
graphene hybrid can be prepared by the direct assembly of hemin
on graphene with p–p interaction.4a Interestingly, graphene oxide
(GO) cannot improve the catalytic activity of hemin due to its
damaged structure.4d Through the chemical reduction of GO, the
enhancement properties of graphene can be recovered.5 However,
the low water-solubility of graphene or reduced GO and the
irreversible assembly of hemin limit its application in bioanalysis.
DNA-linked hemin can provide a feasible way to control the
attachment/detachment of hemin on the GO surface according
to the different affinities of GO toward single-stranded DNA (ssDNA)
and double-stranded DNA (dsDNA).6 Furthermore, hemin can
catalyze a non-fluorescent substrate to form a fluorescent
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry
and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China.
Scheme 1 Schematic illustration of (A) inhibition effect of GO toward
peroxidase activity of ssDNA- or dsDNA-linked hemin, and (B) homogenous
fluorescence strategy for DNA detection.
E-mail: hxju@nju.edu.cn; Fax: +86 25 83593593; Tel: +86 25 83593593
† Electronic supplementary information (ESI) available: Experimental details and
additional figures. See DOI: 10.1039/c4cc02273d
6714 | Chem. Commun., 2014, 50, 6714--6717
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fluorescent 20,70-dichlorofluorescein in the presence of H2O2
(Scheme S1B, ESI†). Although the catalytic fluorescent signal was also
inhibited by GO, the inhibiting efficiency of 57.1% was lower than in
the case of tyramine even at a 20 times higher GO concentration of
15 mg mLꢀ1 (Fig. S3, ESI†). The different inhibition effects of GO
could be explained as shown in Scheme S2 (ESI†). After GO absorbed
the hemin labelled ssDNA probe through p–p interaction between
ssDNA and GO, hemin would be brought to the hydrophobic
p-conjugated carbon structures in the center of GO.9 Compared
with tyramine, H2DCFDA has a larger p-conjugated structure
with more hydrophobic property, which allowed easier access
to hemin absorbed on the GO surface. Thus GO exhibited a
more remarkable inhibition effect for tyramine than H2DCFDA.
The inhibiting efficiency of tyramine depended on the pH of
Fig. 1 (A) Fluorescent spectra of oxidation product of tyramine catalyzed by
10 nM hemin (a), probe (b), probe/GO (c) for 10 min, and probe for 10 min
following by mixing with 0.75 mg mLꢀ1 GO (d). (B) Effect of GO concentration on
its inhibiting efficiency. The catalytic reaction is carried out in 50 mM Tris-HCl
(pH 7.4, containing 300 mM NaCl) with 0.7 mM tyramine and 2 mM H2O2.
reversible, and could be relieved by hybridizing the probe the reaction buffer. Although the inhibiting efficiency increased
with a complementary DNA target to detach the formed dsDNA with decreasing pH from 9.0 to 6.5 (Fig. 2A), the probe showed
and thus the linked hemin from the GO surface. Based on this the highest catalytic fluorescent signal at pH 7.4 (Fig. S4, ESI†).
discrimination, a homogenous fluorescence DNA sensing strategy Thus pH 7.4 was chosen as the optimal value for the next experi-
with high sensitivity was conveniently developed (Scheme 1B).
ments. The inhibiting efficiency on catalytic fluorescent signal also
To verify the feasibility of the proposed method, tyramine was depended on the salt concentration of the buffer. As shown in
used as a non-fluorescent substrate, which could be catalytically Fig. 2B, with the increasing NaCl concentration, the inhibiting
oxidized into a fluorescent dimeric phenol product via the perox- efficiency increased and reached a maximum at 300 mM. A higher
idase activity of the probe in the presence of H2O2 (Scheme S1A, concentration of Clꢀ led to the axial coordination between Fe3+ and
ESI†). Compared with the weak fluorescence (FL) signal catalyzed by Clꢀ, and thus slightly lowered peroxidase activity of hemin. Both
free hemin (Fig. 1A, curve a), the fluorescence signal catalyzed by the low pH and high NaCl concentration were in favour of minimiza-
probe was 7.3 times higher (Fig. 1A, curve b). This phenomenon tion of the electrostatic repulsion between the negatively charged
could be attributed to the fact that hemin possesses weak solubility probe and GO, thus resulting in the closer contact between labeled
in aqueous solution, which led to its self-aggregation and lowered hemin and GO as well as intense peroxidase activity inhibition by
the peroxidase activity,4a while the grafting of hemin with ssDNA GO. High concentrations of tyramine and H2O2 slightly decreased
improved its solubility and thus excluded the aggregation to the inhibiting efficiency due to the limited amount of probe/GO
produce high peroxidase activity.8 Further, when the probe was and the maximum efficiency was achieved at 0.7 mM tyramine and
absorbed onto the GO surface, the catalytic fluorescence signal was 2 mM H2O2 (Fig. 2C and D).
significantly suppressed with a decrease of 88.5% (Fig. 1A, curve c).
The catalytic property of the hemin labeled ssDNA probe was
The signal inhibition could be attributed to two reasons that are as further investigated by recording fluorescence kinetic curves.
follows: GO possessed a strong quenching effect on the fluorescence After adding tyramine and H2O2 to initiate the catalytic reaction,
of the catalyzed product, and the peroxidase activity of hemin was
inhibited after it was absorbed on GO. To clarify this phenomenon,
a control experiment was carried out by adding GO to the product of
the catalytic reaction. In this case, the decrease of fluorescence
intensity was attributed to only the fluorescent quenching effect of
GO, which showed a signal decrease of 11.5% (Fig. 1A, curve d).
Therefore, the inhibition of peroxidase activity of hemin by GO
in this system was mainly responsible for the decrease of the
fluorescent signal. The inhibition effect could be attributed to the
decreasing diffusion rate of the probe after absorption on GO,
the steric hindrance effect of GO and the possible interaction of GO
with the oxidation intermediate. The latter could be concluded by
the effect of GO concentration on its inhibiting efficiency. With the
increasing GO concentration, the inhibiting efficiency increased
and reached a plateau at 0.75 mg mLꢀ1 (Fig. 1B), indicating that the
peroxidase activity of DNA-linked hemin could be regulated by
changing the GO concentration.
To clarify the inhibition efficiency of GO toward the peroxidase
activity of the probe, the 20,70-dichlorodihydrofluorescein diacetate
Fig. 2 Effects of (A) pH, (B) NaCl, (C) tyramine and (D) H2O2 concentration
on inhibiting efficiency of GO toward fluorescent signal of oxidation
(H2DCFDA) as the non-fluorescent substrate was involved in
this system. This compound could be catalytically oxidized into
product catalyzed by the probe. The concentration of probe and GO is
10 nM and 0.75 mg mLꢀ1, respectively.
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Under the optimized conditions, the FL intensity was propor-
tional to the target concentration ranging from 0.5 to 40 nM
(Fig. 3B). The linear regression equation was I = 50.6 + 4.40 ꢁ c
(R = 0.9985), where I is the FL intensity, and c is the target
concentration. The detection limit was estimated at 3s, which
was calculated in the absence of the target for five parallel
detections, to be 0.2 nM. The detection limit was 60 times lower
than that in our previous work using GO and CdTe QDs as the
fluorescent quencher and emitter for DNA sensing,11 and also
lower than those of previously reported homogeneous fluores-
cence DNA sensing strategies using GO or other nanomaterials
as fluorescent quenchers (Table S2, ESI†).12 The specificity of
the protocol was demonstrated to be acceptable for discrimi-
nating the single- and three-base mismatched DNA from the
Fig. 3 (A) Fluorescence kinetic curves of the catalytic reaction for 10 nM
probe (a), probe/target duplex (b), probe/GO (c) and probe/target/GO at
target concentrations of 10 (d), 20 (e) and 40 nM (f). (B) Fluorescent spectra
at 0, 0.5, 1.3, 2.5, 5.0, 10, 20, 30 and 40 nM target (from bottom to top).
Inset: calibration curve.
the fluorescent signal of the oxidation product catalyzed by complementary target (Fig. S7, ESI†).
probe or probe/target duplex increased obviously and reached a
This work identified the inhibition effect of GO toward the
plateau at 10 minutes (Fig. 3A, curves a and b), indicating that peroxidase activity of DNA-linked hemin and developed a simple
hybridization with the target would barely affect the peroxidase homogenous fluorescence strategy for the highly sensitive detection of
activity of the probe. When GO was introduced to interact with DNA. The inhibition effect of GO toward the peroxidase activity of
the probe, the fluorescent signal catalyzed by probe/GO was greatly DNA-linked hemin could be mainly attributed to the decreasing
suppressed (Fig. 3A, curve c). However, when the probe was first diffusion rate of the probe, the steric hindrance of GO, the interaction
hybridized with target DNA from 10 to 40 nM and then GO was of GO with the oxidation intermediate, and the hydrophobicity of
added, the fluorescence signal catalyzed by the probe enhanced p-conjugated GO center. The tunable peroxidase activity of DNA-
gradually (Fig. 3A, curves d–f). This change could be attributed to the linked hemin was realized by hybridizing the probe with target
weaker affinity of GO to dsDNA than ssDNA due to the rigid duplex DNA, which led to a homogeneous fluorescence DNA sensing strategy
structure and the shield of the phosphate backbone on the nucleo- with high sensitivity and acceptable specificity. This catalytic fluores-
tide bases, thus weakening the interaction between hemin labelled cence strategy was simple, relatively quick, and could be conveniently
dsDNA and GO, and the inhibition effect of GO.
combined with other signal amplification steps for further improving
The interaction between the probe or probe/target duplex the sensitivity. Also, the proposed inhibition effect could be easily
and GO was demonstrated using gel electrophoresis. As shown extended to design other analytical applications.
in Fig. S5 (ESI†), the probe displayed only one band in the gel
This research was financially supported by the National Basic
(lane a). After interaction with GO, the probe/GO did not show Research Program of China (2010CB732400), and the National
an obvious band (lane b) because the probe was absorbed by GO Natural Science Foundation of China (21135002, 21121091).
to form the probe/GO, which could hardly penetrate into the gel
due to the large steric hindrance of GO.10 However, when the
probe was first hybridized with target and then mixed with GO,
the probe/target duplex exhibited a slow-moving band (lane c),
suggesting that the hybridization of the target with probe led to
the detachment of the probe from GO.
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The different inhibition effects of GO toward the peroxidase
activity of ssDNA- and dsDNA-linked hemin offered an opportunity
to design a novel homogeneous fluorescence DNA sensing strategy.
GO was usually used as fluorescence quencher in conventional
fluorescence DNA sensing strategies,6 and theoretically one target
DNA could only recover the fluorescence of one fluorophore labeled
on the probe (Scheme S3, ESI†). In our strategy, GO was utilized to
inhibit the peroxidase activity of hemin labeled on the probe, thus
theoretically one target DNA could recover the peroxidase activity of
one hemin, and then hemin could catalyze the reaction of dozens of
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and target was optimized to be 10 min (Fig. S6A, ESI†), and the GO
concentration was optimized to be 0.75 mg mLꢀ1 (Fig. S6B, ESI†).
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