Peroxidase-mimicking DNAzyme modulated growth of CdS nanocrystalline structures in situ through redox reaction: Application to development of genosensors and aptasensors

Article abstract of DOI:10.1021/ac502360y

This work demonstrates the use of the peroxidase-mimicking DNAzyme (peroxidase-DNAzyme) as general and inexpensive platform for development of fluorogenic assays that do not require organic fluorophores. The system is based on the affinity interaction between the peroxidase-DNAzyme bearing hairpin sequence and the analyte (DNA or low molecular weight molecule), which changes the folding of the hairpin structure and consequently the activity of peroxidase-DNAzyme. Hence, in the presence of the analyte the peroxidase-DNAzyme structure is disrupted and does not catalyze the aerobic oxidation of l-cysteine to cystine. Thus, l-cysteine is not removed from the system and the fluorescence of the assay increases due to the in situ formation of fluorescent CdS nanocrystals. The capability of the system as a platform for fluorogenic assays was demonstrated through designing model geno- and aptasensor for the detection of a tumor marker DNA and a low molecular weight analyte, adenosine 5triphosphate (ATP), respectively.

Full text of DOI:10.1021/ac502360y

Article
pubs.acs.org/ac
Peroxidase-Mimicking DNAzyme Modulated Growth of CdS
Nanocrystalline Structures in Situ through Redox Reaction:
Application to Development of Genosensors and Aptasensors
̈
Gaizka Garai-Ibabe, Marco Moller, Laura Saa, Ruta Grinyte, and Valeri Pavlov*
Biofunctional Nanomaterials Unit, CIC BiomaGUNE, Parque Tecnolog
́ ́
ico de San Sebastian, Paseo Miramon 182, Donostia-San
Sebastian, 20009, Spain
́
*
S Supporting Information
ABSTRACT: This work demonstrates the use of the peroxidase-
mimicking DNAzyme (peroxidase-DNAzyme) as general and
inexpensive platform for development of fluorogenic assays that do
not require organic fluorophores. The system is based on the affinity
interaction between the peroxidase-DNAzyme bearing hairpin
sequence and the analyte (DNA or low molecular weight molecule),
which changes the folding of the hairpin structure and consequently
the activity of peroxidase-DNAzyme. Hence, in the presence of the
analyte the peroxidase-DNAzyme structure is disrupted and does not
catalyze the aerobic oxidation of L-cysteine to cystine. Thus, L-cysteine
is not removed from the system and the fluorescence of the assay
increases due to the in situ formation of fluorescent CdS nanocrystals. The capability of the system as a platform for fluorogenic
assays was demonstrated through designing model geno- and aptasensor for the detection of a tumor marker DNA and a low
molecular weight analyte, adenosine 5′triphosphate (ATP), respectively.
he DNAzymes are nucleic acids with catalytic activity,
DNA complexes have been used together with presynthesized
T
isolated from combinatorial oligonucleotide libraries by in
fluorescent QDs for the detection DNA, low molecular weight
1
12,13
vitro selection. Since the first DNAzyme was reported in 1994,
many other DNAzymes with different catalytic activities have
analytes, and telomerase activity.
Semiconductor QDs are
extensively used as labels for the optical and electrochemical
1
,2
14
been described.
In comparison to conventional proteic
sensing of biorecognition events. They can be photoexcited to
enzymes, DNAzymes show clear advantages including higher
stability and they can be denatured and renatured without
losing catalytic activity. Their synthesis is cost-effective, and
they can be massively produced by a DNA synthesizer with
desirable modifications. These unique characteristics make
DNAzymes ideal biocatalysts to be applied for development of
biosensors.
generate electron/hole couples which recombine to yield
15
fluorescent emission of light. Furthermore, their physico-
chemical properties are defined by their submicrometer
dimensions and differ significantly from those of the
3
16
corresponding bulk material. The advantages of QDs over
traditional organic fluorophores, broadly used in detection of
DNA, include higher quantum yield, reduced photobleaching,
17
Application of peroxidase-mimicking DNAzymes (perox-
idase-DNAzyme) to bioanalysis was pioneered by the group of
and higher extinction coefficient. However, all reported assays
were based on the conjugation between presynthesized QDs
1
2,13
4
,5
and peroxidase-DNAzyme.
Usually, they suffer from
Itamar Willner. The common structural unit of such DNA
showing peroxidase activity is a guanine-rich sequence that in
the presence of hemin forms a catalytically active hemin/G-
quadruplex complex. This hemin−DNA complex catalyzes
oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonica-
cid) (ABTS ) and luminol with H O to yield colored product
ABTS and chemoluminescent oxidation products, respec-
tively.
insufficient quenching of QDs by organic quenchers, non-
specific adsorption on surfaces, and require complicated
synthetic procedures to produce semiconductor QDs employ-
ing dangerous materials like Te.
2−
Different unconventional routes have been introduced by our
group to achieve in situ biocatalytical growth of CdS
nanocrystals. It was demonstrated that S2 ions generated
through enzymatic reactions interact with exogenously added
2
2
•−
−
5
−7
The oxidation of nonfluorescent precursors to
8
,9
fluorescent reporter molecules was also reported. These
catalytic activities were extensively used in bioanalytical assays
to transduce and amplify the read-out signal of biorecognition
events, such as DNA hybridization or aptamer−analyte
2+
18,19
Cd to yield CdS QDs.
Enzymes were also used to
produce QDs capping agents, such as thiolated products
5
,8,10
complexation.
The group of Evgenii Katz pioneered
Received: March 5, 2014
application of hemin−DNA complexes to biocomputing
Accepted: September 17, 2014
11
security systems and biocatalytical multiplexers. Hemin−
©
XXXX American Chemical Society
A
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(
reduced glutathione and thiocholine) and orthophosphate that
concentrations of 3. After 30 min of incubation at RT, 3 ×
20−22
−6
were able to affect the growth of fluorescent QDs.
should be noted that these systems were successfully applied for
the determination of clinically relevant enzymatic activities and
It
10 M Sn(IV) protoporphyrin IX was added and we let them
−4
interact for 20 min at RT. Afterward, 2 × 10 M L-cysteine was
added to the assay, and the mixture was incubated for 10 min at
1
8−21,23
−4
inhibitors.
RT, followed by the addition of 2.5 × 10 M Na S and 1.25 ×
2
−3
Recently, a novel catalytic activity of the peroxidase-
DNAzyme was reported: the aerobic oxidation of thiols to
disulfides in the absence of exogenously added H O . The
10 M Cd(NO ) necessary for the in situ formation of CdS
3 2
QDs. The mixture was incubated for 5 min at RT, and the
fluorescence emission spectra of the resulting suspension were
recorded at λexc = 260 nm.
9
2
2
present study reports on the use of this catalytic activity as a
general and inexpensive platform for the development of label-
free and enzyme-free homogeneous fluorogenic assays. For the
first time the DNAzyme catalytic activity was used to modulate
the fluorescence of the assay, through the control of the growth
of CdS crystals. Furthermore, to prove the potential of the
system for biosensors development, model fluorogenic
genosensor and aptasensors were created for the detection of
a tumor marker DNA and adenosine 5′-triphosphate.
ATP Detection by the Peroxidase-DNAzyme Modu-
lation of in Situ Growth CdS QDs. In a typical experiment 2
−6
× 10 M 6 was incubated in HEPES buffer 5 mM containing
10 mM NaNO3 (pH 7.5) in the presence of variable
concentrations of ATP. After 30 min of incubation at RT, 3
−
6
×
10 M Sn(IV) protoporphyrin IX was added and we let
−4
them interact for 20 min at RT. Afterward, 2 × 10 M L-
cysteine was added to the assay, and the mixture was incubated
−
4
for 10 min at RT, followed by the addition of 2.5 × 10
M
−3
EXPERIMENTAL SECTION
Na S and 1.25 × 10 M Cd(NO ) necessary for the in situ
■
2
3 2
Materials. Adenosine 5′ diphosphate (ADP) and proto-
formation of CdS QDs. The mixture was incubated for 5 min at
RT, and the fluorescence emission spectra of the resulting
suspension were recorded at λexc = 260 nm.
3
+
3+
4+
2+
porphyrin IX containing different ions (Co , Mn , Sn , Zn ,
2+
2+
Cu , Mg ) were purchased from Acros Organics (Belgium)
and Frontier Scientific (Utah, U.S.A.), respectively. Adenosine
Transmission Electron Microscopy Study. For CdS
QDs characterization by transmission electron microscopy
(TEM), L-cysteine-stabilized CdS QDs were produced in
5
′ triphosphate (ATP), uridine 5′-triphosphate (UTP), cytidine
5
′-triphosphate (CTP), and guanosine 5′-triphopsphate
(
GTP), dimethyl sulfoxide (DMSO), hemin, and other
HEPES buffer 5 mM containing 10 mM NaNO (pH 7.5) in
3
−
4
−4
chemicals were obtained from Sigma-Aldrich (Spain) and
used as supplied. Sn(IV) protoporphyrin IX solutions in
DMSO (5 mM) were freshly prepared prior to each
experiment. The oligonucleotide sequences used in this study
were synthesized by Integrated DNA Technologies (IDT,
Belgium): 1, 5′-TTTGGGTAGGGCGGGTTGGG-3′; 2, 5′-
GGGATGGGTCGCAAAGAACGAGAGTCTTTCTGCGA-
GGGTTGGG-3′; 3, 5′-CAGAAAGACTCTCGTTCTTT-3′;
the presence of 2 × 10 M L-cysteine, 2.5 × 10 M Na S, and
2
1.25 × 10− M Cd(NO ) . Such prepared samples were freshly
3
3
2
proceeded by desiccating a droplet of the solution on surfaces
of hydrophilized, ultrathin carbon film coated copper grids or
thin silicon oxide or silicon nitride membranes. Data was
acquired in a TEM of type JEM-2100F-UHR (JEOL, Japan)
equipped with a high-angle annular dark field (HAADF)
detector, a digital camera of type F-216 (TVIPS, Germany),
and an energy-dispersive X-ray spectroscopy (EDXS) system of
type INCA (Oxford, U.K.).
4
, 5′-CAGAAAGACTCTCCTTCTTT-3′; 5, 5′-CAGAAAGA-
GTGTCGTTCTTT-3′; 6, 5′-GGGATGGGCACCTGGGG-
GAGTATTAGCGGAGGAAGGTTCCAGGTGGGGTTG-
GG-3′.
RESULTS AND DISCUSSION
■
Instruments. All assays were performed in black flat-well
Scheme 1 illustrates the reported system. Briefly, the active
peroxidase-DNAzyme (1), Sn(IV) protoporphyrin IX/perox-
idase-DNAzyme complex, catalyzes the oxidation of the
(
330 μL) NUNC 96 wells microtiter plates, and the
fluorescence spectra were recorded with a Varioskan Flash
fluorimeter (Thermo Scientific). All water used was Milli-Q
ultrapure grade (EMD Millipore).
Peroxidase-DNAzyme Catalyzed Oxidation of L-Cys-
teine to Cystine and Modulation of the Assay
Fluorescence. To prove the operation mechanism of the
proposed system control experiments were performed. Prior to
Scheme 1. Oxidation of L-Cysteine to Cystine by Active
Peroxidase-DNAzyme Results in Modulation of QD Growth
the experiments oligonucleotides were dissolved in 10 mM
phosphate buffer (pH 7.4). In a typical experiment 2 × 10−
6
M
1
was incubated in HEPES buffer 5 mM containing 10 mM
NaNO (pH 7.5) in the presence of 3 × 10 M Sn(IV)
−6
3
protoporphyrin IX for 20 min at room temperature (RT).
Afterward, 2 × 10− M L-cysteine was added to the assay, and
the mixture was incubated for 10 min at RT, followed by the
4
−4
−3
addition of 2.5 × 10 M Na S and 1.25 × 10 M Cd(NO3)2
2
necessary for the in situ formation of CdS QDs. The mixture
was incubated for 5 min at RT, and the fluorescence emission
spectra of the resulting suspension were recorded at λexc = 260
nm.
DNA Detection by the Peroxidase-DNAzyme Modu-
lation of in Situ Growth CdS QDs. In a typical experiment, 2
−
6
×
1
10 M 2 was incubated in HEPES buffer 5 mM containing
0 mM NaNO3 (pH 7.5) in the presence of variable
B
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thiolated molecule (L-cysteine) to its corresponding disulfide
intrinsic catalytic activity of Sn(IV) protoporphyrin IX.
However, when both Sn(IV) protoporphyrin IX and
peroxidase-DNAzyme were present in the system, the complex
Sn(IV) protoporphyrin IX/peroxidase-DNAzyme was formed
to catalyze the oxidation of thiol to disulfide. As a consequence,
a detectable reduction of the fluorescent signal was recorded
due to the depletion of the L-cysteine necessary for the
stabilization of the in situ growing CdS QDs. To further
demonstrate that the fluorescent signal was caused by the in
situ formation of CdS QDs, experiments were performed in the
(
cystine). It should be noted that the H O required for the
2 2
oxidation of L-cysteine is not exogenously added, as it is
autocatalytically generated from O during the disulfide
2
9
formation. The formed cystine cannot bind to the surface of
growing CdS crystals generated via the interaction of Cd with
2
+
2
−
S , due to the lack of the mercapto group. Thus, the oxidation
of L-cysteine to cystine results in the decrease of the
fluorescence of the assay, as cystine has low capacity to
stabilize the growth of fluorescent CdS QDs. It should be noted
that, in order to optimize the peroxidase-DNAzyme-catalyzed
thiol oxidation, a number of protoporphyrin IX molecules
absence of Cd(NO ) , the metallic salt required for the
3 2
formation of QDs (Figure 1, curve e), and in the absence of
3
+
3+
3+
4+
2+
Cd(NO3) and Na S (Figure 1, curve f). Thus, we
containing different metal ions (Fe , Co , Mn , Sn , Zn ,
2
2
2
+
2+
demonstrated that the peroxidase-DNAzyme catalyzing thiol
oxidation could be used to modulate the fluorescent readout
signal, through the consumption of the stabilizing thiolated
agent. An affinity interaction between the peroxidase-
DNAzyme bearing molecular hairpin and the corresponding
analyte is able to change the peroxidase activity of the
DNAzyme.
In order to characterize the cysteine-stabilized CdS QDs,
UV−visible (UV−vis) absorption and emission spectra were
recorded in buffer solution (solid and dashed lines,
respectively) as showed in Figure 2A. From the UV−vis
Cu , Mg ) were complexed with the peroxidase-DNAzyme.
Afterward, the catalytic activity of the formed metal
protoporphyrin IX/peroxidase-DNAzyme complexes was tested
in terms of the fluorescent signal reduction capacity, as
consequence of the depletion of the stabilizing agent (L-
cysteine) and the disulfide (cystine) formation (Supporting
Information Table S1). Taking into consideration these results,
Sn(IV) protoporphyrin IX was selected for subsequent studies,
instead of the commonly used hemin (Fe(III) protoporphyrin
IX). CdS QDs formation time of 5 min was selected as the
optimal incubation time to standardize the time period to
perform reproducible fluorescent measurements. No further
growth in fluorescence intensity was observed after 5 min of
2
+
2−
incubation. Optimum concentrations of Cd and S were
found by comparing fluorescent signals arising from CdS QDs
produced in assay mixtures containing inactive DNAzyme
(DNAzyme only) and active DNAzyme (DNAzyme recon-
stituted with Sn(IV) protoporphyrin) (Supporting Information
Table S2). The maximum ratio between fluorescence signals
2
+
2−
was observed at [Cd ] = 1.25 mM and [S ] = 0.25 mM;
therefore, those concentrations were used for the subsequent
experiments.
In order to attest the operating mechanism of our system, a
number of control experiments were carried out (Figure 1).
The highest emission peak attributed to QDs grown in situ was
observed in the absence of Sn(IV) protoporphyrin IX in the
reaction mixture (Figure 1, curves a and b). When only Sn(IV)
protoporphyrin IX was present in the system, the fluorescent
emission intensity was reduced (Figure 1, curve c) due to the
Figure 2. (A) Absorption (solid line) and emission (dashed line) of
the formed CdS nanocrystals. (B) HRTEM image of CdS nanocryst-
als.
spectrum, we observed an increased absorption below 500 nm
and a shoulder at about 350 nm. The presence of this shoulder
is explained by the excitonic transition between the electron 1S
state and the hole 1S state, in semiconductor nanocrystals with
24
a diameter about 3 nm. On the other hand, the emission
spectrum demonstrates a well-shaped peak at 550 nm which
arises from excitonic emission of CdS nanocrystals. Further-
more, TEM was used to confirm the existence of stable CdS
nanocrystals in the reaction mixture. Previously, it was reported
in the literature that CdS nanoparticles produced from aqueous
solutions using L-cysteine as stabilizer form aggregates on TEM
25
grids, contrary to separate nanoparticles produced with other
1
9,20
stabilizing agents.
So the morphology and size of such CdS
nanocrystals were evaluated on the basis of high-resolution
TEM (HRTEM) images of CdS aggregates as shown in Figure
2
B. The TEM-EDXS study on the produced material proves it
Figure 1. Fluorescence emission spectra of CdS QDs formed in (a)
the absence of peroxidase-DNAzyme and Sn(IV) protoporphyrin IX,
to be composed of Cd and S in a 1:1 ratio. Images taken at
STEM-HAADF and HRTEM conditions endorse that CdS
nanocrystals of hexagonal phase have been formed (Supporting
Information Figure S1).
(
(
b) peroxidase-DNAzyme only, (c) Sn(IV) protoporphyrin IX only,
d) the presence of peroxidase-DNAzyme and Sn(IV) protoporphyrin
IX, (e) absence of Cd(NO ) , (f) absence of Cd(NO ) and Na S.
3
2
3
2
2
C
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Scheme 2. DNA Detection through Peroxidase-DNAzyme Modulated Growth of CdS QDs in Situ
Scheme 2 shows the proposed system as a general platform
for genosensors development based on the peroxidase-
DNAzyme catalytic activity-mediated fluorescence intensity
modulation. Briefly, a hairpin (2) consisting of three parts was
designed: a single-stranded loop domain, complementary to the
analyte DNA (green), a self-complementary sequence that
stabilized the structure (pink), and the peroxidase-DNAzyme
sequence (blue), split in two halves, placed at both ends of the
construction. The free hairpin structure, in the absence of the
target DNA and in the presence of Sn(IV) protoporphyrin IX,
forms the peroxidase-DNAzyme sequences which self-assemble
into functional Sn(IV) protoporphyrin IX/peroxidase-DNA-
zyme complexes to catalyze the oxidation of L-cysteine to
cystine. The depletion of the thiol-bearing molecule (L-
cysteine), required for the stabilization of the growing CdS
crystals to form fluorescent CdS QD, results in inhibition of
their growth and low fluorescent signal. On the other hand,
when the analyte DNA is present in the assay, the affinity
interaction between the loop domain and the target DNA
disrupts the structure of the hairpin and the Sn(IV)
protoporphyrin IX/peroxidase-DNAzyme complex cannot be
formed in the presence of protoporphyrin IX. As a
consequence, L-cysteine is not converted into cystine and
binds to the surface of CdS crystals facilitating the growth of
fluorescent NPs. Thus, the fluorescence intensity of the assay
increases with the increase in concentration of the analyte
DNA.
Figure 3. Fluorescence emission spectra of CdS QDs formed in the
presence of variable concentrations of the DNA species related to
−
7
−7
ORAOV1 gene: (a) 0 M, (b) 1 × 10 M, (c) 2.5 × 10 M, (d) 5 ×
−7
−6
−6
−6
1
1
0
0
M, (e) 1 × 10 M, (f) 2.5 × 10 M, (g) 5 × 10 M, (h) 1 ×
−5
M.
present in the system the switching of the molecular hairpin
structure reduces the number of Sn(IV) protoporphyrin IX/
peroxidase-DNAzyme complexes in the assay. Thus, L-cysteine
is not removed from the system and can stabilize in situ
growing CdS QDs to increase the detected fluorescence. The
calibration curve (Figure 3B) was generated by plotting the
fluorescence intensity, at 510 nm, generated by the in situ
produced CdS QDs in the presence of variable concentrations
of the tumor marker DNA. One can see that the system allows
the analysis of the target DNA with a limit of detection of 100
nM, which is in the range of previously developed methods for
DNA detection using enzymatic amplification free catalytic
To prove the suitability of the proposed system for the
detection of DNA, a model genosensor was developed for the
detection of a DNA species related to oral cancer overexpressed
1
gene (ORAOV1) (3). ORAOV1 gene is overexpressed in
5,27
many solid tumors and this fact makes it a potential candidate
molecular hairpins.
In addition, the specificity of the
2
6
as tumor marker. Figure 3A shows the fluorescence spectra
obtained in the presence of 2 μM of the peroxidase-DNAzyme
bearing molecular hairpin and different concentrations of the
target tumor marker DNA. As can be seen, when the
concentration of the analyte DNA increases, the fluorescence
of the system increases because of the higher concentration of
L-cysteine in the assay. Hence, when the analyte DNA is
proposed system was tested using one- (4) and two-base (5)
mismatched target DNA sequences. Results showed that the
system is specific only for the target DNA, and the presence of
mismatched target DNA sequences (10 μM) yields a low
fluorescence signal close to the background fluorescence of the
system (Supporting Information Figure S2). Thus, we believe
that with the development of this model system for the
D
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Article
detection of a DNA sequence related with ORAOV1 gene, we
demonstrate its capability as a general platform for genosensors
development.
The peroxidase-DNAzyme catalytic activity mediated mod-
ulation of the in situ generation of QDs, was also applied to the
development of a model fluorogenic aptasensors for low
molecular weight analyte detection. The system was applied to
the detection of ATP as depicted in Scheme 3. A catalytic DNA
Scheme 3. ATP Detection through Peroxidase-DNAzyme
Modulated Growth of CdS QDs in Situ
Figure 4. Fluorescence emission spectra of CdS QDs formed in the
−6
presence of variable concentrations of ATP: (a) 0 M, (b) 5 × 10 M,
−
5
−5
−5
−4
(
(
c) 1 × 10 M, (d) 2.5 × 10 M, (e) 5 × 10 M, (f) 1 × 10 M,
4
−
g) 2.5 × 10 M.
concentration of adenosine diphosphate (ADP), uridine 5′-
triphosphate (UTP), cytidine 5′-triphosphate (CTP), and
guanosine 5′-triphopsphate (GTP) in the assay mixture.
ADP, UTP, CTP, and GTP did not show any affinity
interaction with the aptamer sequence leading to a fluorescent
signal close to the background fluorescence, as demonstrated in
Supporting Information Figure S3. Our system is able to
distinguish between ATP and ADP. Such assay could find
applications in biochemistry, because many biological pathways
involve conversion of ATP to ADP and vice versa.
hairpin was designed (6) consisting in an ATP aptamer
1
3
sequence (green), a self-complementary sequence that
stabilized the structure (pink), and the peroxidase-DNAzyme
sequence (blue), split in two halves, placed at both ends of the
oligonucleotide. In the absence of the analyte and in the
presence of Sn(IV) protoporphyrin IX, the sequence folds into
the catalytically active peroxidase-DNAzyme structure to
oxidize L-cysteine to cystine, which decreases the fluorescence
of the assay as stated above. When ATP is present in the
system, the affinity interaction between ATP and the aptamer
sequence results in the disruption of the hairpin structure and
the peroxidase-DNAzyme sequence cannot fold into the active
conformation of peroxidase-DNAzyme even in the presence of
Sn(IV) protoporphyrin IX. As L-cysteine is not removed from
the assay solution, fluorescent CdS QDs can be formed via the
stabilization of the in situ growing CdS nanocrystals with the
thiolated product, enabling the fluorimetric detection of ATP
molecules.
CONCLUSIONS
■
In this work we demonstrated the application of the peroxidase-
DNAzyme catalyzed thiol oxidation to disulfides for the
development of general and inexpensive fluorimetric geno-
and aptasensors, based on the active peroxidase-DNAzyme
mediated modulation of in situ produced QDs. It should be
noted that the novelty of the reported sensing strategy lies on
the use of inexpensive compounds, such as DNA and salts, for
the development of fluorimetric bioanalytical systems. In
comparison with other reported fluorogenic assays based on
presynthesized QDs modified with recognition elements, our
assays require neither any synthetic procedures for chemical
modification of semiconductor NPs nor any organic fluorogenic
enzymatic substrates. However, in order to reduce the
background signal, improve the limit of detection, and finally
to improve the signal to background ratio of a prospective
assay, the present system will be coupled with analyte recycling
DNA machinery operating by strand displacement. We believe
that our system can serve as a model for new inexpensive tests
for detection of relevant analytes in clinical diagnosis.
Figure 4A depicts the fluorescent emission spectra upon
analyzing different concentrations of ATP in the presence of a
fixed concentration of the peroxidase-DNAzyme bearing
molecular hairpin. As one can see from the increasing intensity
of the fluorescence peaks, the amount of formed CdS QDs
depends on the ATP concentration. Thus, the increase in ATP
concentration leads to the decrease in the number of active
peroxidase-DNAzyme molecules, and hence, the fluorescence
intensity in the assay mixture grows. Figure 4B shows the
derived calibration curve corresponding to the fluorescence
intensity, at 510 nm, generated in the assay mixture in the
presence of different concentrations of ATP. According to the
calibration plots the developed ATP detection system allows
the detection of 5 μM of ATP, which is in the range of
previously developed methods for ATP detection using
ASSOCIATED CONTENT
Supporting Information
■
*
S
TEM analysis of the CdS nanostructures, analysis of the
influence of metal ions on the catalytic activity of
protoporphyrin IX, and control experiments for DNA and
ATP detection. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +34 943005308. Fax: +34 943005314. E-mail:
vpavlov@cicbiomagune.es.
■
2
7,28
enzymatic amplification free catalytic molecular hairpins.
To test the specificity of the reported system, the variation of
the fluorescence intensity was tested in the presence of high
E
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Analytical Chemistry
Article
Notes
1779−1786. (b) Zhai, C.; Li, Y.; Mascarenhas, C.; Lin, Q.; Li, K.;
Vyrides, I.; Grant, C. M.; Panaretou, B. Oncogene 2014, 33, 484−494.
The authors declare no competing financial interest.
(
27) Zhang, Z.; Sharon, E.; Freeman, R.; Liu, X.; Willner, I. Anal.
Chem. 2012, 84, 4789−4797.
28) Huang, J.; He, Y.; Yang, X.; Wang, K.; Quan, K.; Lin, X. Analyst
014, 139, 2994−2997.
ACKNOWLEDGMENTS
■
(
2
This work was supported by the Spanish Ministry of Economy
and Competitiveness (project BIO2011-26356) and the
ETORTEK program from the Department of Economic
Development and Competitiveness from the Basque Govern-
ment.
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