sensors
Article
A Sensitive and Label-Free Pb(II) Fluorescence
Sensor Based on a DNAzyme Controlled
G-Quadruplex/Thioflavin T Conformation
Yanli Wen, Lele Wang, Lanying Li, Li Xu and Gang Liu *
Biometrology Laboratory, Division of Chemistry and Ionizing Radiation Measurement Technology,
Shanghai Institute of Measurement and Testing Technology, Shanghai 201203, China;
[email protected] (Y.W.); [email protected] (L.W.); [email protected] (L.L.); [email protected] (L.X.)
* Correspondence: [email protected]; Tel.: +86-21-3883-9800 (ext. 35324)
Academic Editor: Huangxian Ju
Received: 3 October 2016; Accepted: 24 November 2016; Published: 16 December 2016
Abstract: Pb(II) can cause serious damaging effects to human health, and thus, the study of Pb2+
detection methods to sensitively and selectively monitor Pb(II) pollution has significant importance.
In this work, we have developed a label-free fluorescence sensing strategy based on a Pb(II) DNAzyme
cleavage and the ThT/G-quadruplex complex. In the presence of Pb(II), a G-rich tail was cut and
released from the substrate strand, which then would form a G-quadruplex structure by combination
with ThT dye. The fluorescence signal increase was then measured for sensitive Pb(II) quantification
with a limit of detection of 0.06 nM. Our sensor also demonstrated high selectivity against six different
metal ions, which is very important for the analysis of complex samples.
Keywords: DNAzyme; heavy metal ions; lead ion; thioflavin T (ThT)
1. Introduction
Many heavy metal ions such as lead, mercury, arsenic and chromium are highly toxic elements
which can cause a number of serious threats to human health and the environment [1–5]. Among all
the metal ions, Pb2+ is one of the four metals that have the most damaging effects on human health.
It can enter the human body through uptake of food, water and air, and cause serious health problems,
including disruption of the biosynthesis of haemoglobin and anaemia, kidney damage, brain damage,
diminished learning abilities of children and behavioral disruptions of children [4].
In order to protect human health from these threats, the study of sensitive, selective and low-cost
Pb2+ detection methods to monitor lead ion pollution in many fields, including environmental,
water, food safety, etc., has been of significant importance over the past decades. Recently, many
different strategies have been developed toward efficient Pb2+ analysis, including classical atomic
absorption spectrometry (AAS) [6–8], inductively coupled plasma mass spectrometry (ICP-MS) [9,10],
and inductively coupled plasma atomic emission spectrometry (ICP-AES) [11,12]. Unfortunately,
most of these methods require complicated sample pretreatment, multiple analysis steps or expensive
equipment, hindering their application in on-site or real-time analysis.
Metal-specific DNAzymes are a class of well-characterized DNAzymes that cleave an
oligonucleotide substrate containing one ribonucleotide at the cleavage site in the presence of a
particular metal ion [13–17]. As oligonucleotides, high stability DNAzymes can be conveniently
synthesized at low cost and DNAzyme-based sensors have demonstrated a series of advantages
such as fast analysis, nondestructive detection and the capability of providing in situ and real-time
information [18–20], and thus they have been widely recognized as promising candidates for the
development of metal ion sensors. Recently, the research on Pb2+ sensors based on Pb2+ -specific
Sensors 2016, 16, 2155; doi:10.3390/s16122155
www.mdpi.com/journal/sensors
Sensors 2016, 16, 2155
2 of 8
DNAzymes has attracted plenty of research interest. For instance, Lu’s group [3] reported a
colorimetric Pb2+ biosensor based on the DNAzyme-directed assembly of gold nanoparticles.
Xiao et al. [21] developed an electrochemical Pb2+ sensor via an electrode-bound DNAzyme assembly
and achieved part-per-billion (nanomolar) sensitivity. Cropek’s group [22] reported a microchip-based
lead sensor with a lead-specific DNAzyme and fluorescent tags which translated the cleavage events to
measurable, optical signals proportional to Pb2+ concentration. However, most of the fluorescent Pb2+
biosensors need a covalently labeled fluorophore/quencher on the DNAzyme strand, often leading
to complicated synthetic routes, high cost, low synthetic yield and even serious interference with
DNAzyme cleavage [23,24]. Most recently, some groups have reported their inspiring progress in the
development of label-free Pb2+ biosensors, yet the limit of detection (LOD) still remains a challenge for
us [25–28].
Thioflavin T (ThT) is a commercial fluorescent dye, which is capable of binding to the
G-quadruplex structure, generating an increased fluorescence signal, and it has been successfully
utilized in several highly sensitive and label-free fluorescent biosensors [29–32]. In this work, we
designed a novel DNAzyme analysis system by tailing a G-rich sequence onto the substrate strand.
In the presence of Pb2+ , DNAzyme cleaved the substrate strand and released the G-rich part which
subsequently combined with ThT and formed a G-quadruplex structure for an obviously enhanced
fluorescent signal. Our strategy achieved excellent selectivity of Pb2+ over six different metal ions, and
the limit of detection (LOD) of Pb2+ was 0.06 nM and the linear range was from 10 nM to 10 µM Pb2+ .
2. Materials and Methods
2.1. Materials
Pb2+ -specific DNAzyme (Pb-DNAzyme) and the substrate strand DNA (Pb-sub) were synthesized
and purified by TaKaRa Biotech. Co., Ltd. (Dalian, China), and the DNA sequences are shown
as follows:
Pb-DNAzyme:
50 -CCAAAGTGCTCCGAGCCGGTCGAAGTGAAACC-30
Pb-Sub:
50 -GGGTTGGGCGGGATGGGTTTCACTrAGGCACTTTGGGTAGGG-30 , (rA represents an adenosine
ribonucleotide).
Pb(NO3 )2 , Cu(NO3 )2 , Cd(NO3 )2 , Co(NO3 )2 , Mn(NO3 )2 , Ni(NO3 )2 , Hg(NO3 )2 were 1 g/L
certified reference materials (CRMs) obtained from the Shanghai Institute of Measurement and
Testing Technology (Shanghai, China). 4-(2-Hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES)
and 3,6-dimethyl-2-(4-dimethylaminophenyl) benzothiazolium cation (ThT) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). All reagents were of analytical grade and all solutions were
prepared using ultrapure water (18.2 MΩ·cm resistivity).
2.2. Apparatus
Fluorescence spectra were measured on a F-7000 spectrophotometer (Hitachi, Tokyo, Japan)
equipped with a 150 W xenon lamp excitation source, using a quartz cell of 10 mm path length.
The excitation wavelength was 425 nm, and the emission spectrum was from 450 nm to 600 nm.
The slits for excitation and emission were 2.5 nm. A nanodrop 2000 spectrophotometer (Thermo
Scientific, Waltham, MA, USA) was used to quantify the oligonucleotides by measuring the UV
absorbance at 260 nm (OD260).
Sensors 2016, 16, 2155
3 of 8
2.3. DNAzyme-Based Fluorescence Sensor for Pb2+
Pb2+ solutions were prepared in ultrapure water of a series of concentrations, and then 0.3 µM
3 of 8
Pb-DNAzyme, 0.2 µM Pb-sub DNA, 50 mM KCl and 10 mM HEPES buffer were added, and then
the mixture was incubated in 37 ◦ C air bath for 2 h to firstly form the substrate-enzyme complex
DNAzyme and then perform the DNAzyme cleavage. After the cleavage, 20 μM ThT was added to
DNAzyme and then perform the DNAzyme cleavage. After the cleavage, 20 µM ThT was added to
react with the G-rich sequence at 37 °C for 30 min to form the ThT/G-quadruplex complex. Finally,
react with the G-rich sequence at 37 ◦ C for 30 min to form the ThT/G-quadruplex complex. Finally,
the fluorescence signal at 490 nm was measured.
the fluorescence signal at 490 nm was measured.
Sensors 2016, 16, 2155
3. Results
Results
3.
2+ Detection
3.1. The
The Principle
Principle of
of Pb
Pb2+
3.1.
Detection
2+ sensing based on the DNAzyme and the
The label-free
label-free fluorescent
fluorescent strategy
strategy for
for Pb
Pb2+
The
sensing based on the DNAzyme and the
ThT/G-quadruplex complex
ThT/G-quadruplex
complexisisshown
shownschematically
schematicallyininFigure
Figure1.1.The
Thesensing
sensingsystem
systemconsists
consistsofofa
DNAzyme
strand
(Pb-DNAzyme),
a
substrate
strand
(Pb-sub),
and
a
single,
ribo-adenine
a DNAzyme strand (Pb-DNAzyme), a substrate strand (Pb-sub), and a single, ribo-adenine (rA,
(rA,
indicated
as
a
red
point
in
Figure
1)
between
them.
The
substrate
strand
is
tailed
with
a
G-rich
indicated as a red point in Figure 1) between them. The substrate strand is tailed with a G-rich
sequence atatthe
the50 5′
end,
importantly,
of thesequence
G-rich sequence
to the
sequence
end,
andand
mostmost
importantly,
part ofpart
the G-rich
hybridizedhybridized
to the DNAzyme
2+
DNAzyme
strand,the
inhibiting
theof
formation
of G-quadruplex
structure.
In theofpresence
of Pb-sub
Pb ions,
strand,
inhibiting
formation
G-quadruplex
structure. In
the presence
Pb2+ ions,
is
Pb-sub
is
cleaved
at
the
rA
site,
and
thus
released
the
G-rich
strand
to
form
a
G-quadruplex
cleaved at the rA site, and thus released the G-rich strand to form a G-quadruplex structure under
structure
under the
combination
of ThT
dye. Finally,
an obviously
enhanced
signal
the
combination
of ThT
dye. Finally,
an obviously
enhanced
fluorescence
signalfluorescence
was obtained,
and
was
obtained,
and
the
increase
in
the
fluorescence
intensity
was
calculated
as
the
signal
gain
for
2+
the increase in the fluorescence intensity was calculated as the signal gain for Pb quantification
2+ quantification through F-F0, where F0 and F are the fluorescence intensity before and after Pb2+
Pb
2+
through F-F0 , where F0 and F are the fluorescence intensity before and after Pb DNAzyme reaction,
DNAzyme reaction,
respectively.
CDS1)
Spectrum
(Figure S1) and electrophoresis
polyacrylamidegel
electrophoresis
respectively.
CD Spectrum
(Figure
and polyacrylamidegel
results
(Figure S3)
results
(Figure
S3)
demonstrated
the
formation
of
G-quadruplex,
and
when
we
increased
the time
demonstrated the formation of G-quadruplex, and when we increased the time of DNAzyme cleavage,
of
DNAzyme
cleavage,
the
signal
gain
(F-F
0) increased until it reached a plateau at 2 h (Figure S2).
the signal gain (F-F0 ) increased until it reached a plateau at 2 h (Figure S2).
2+
Figure
Figure 1.
1. Illustration
Illustration of
of the
the label-free
label-free fluorescent
fluorescent sensing
sensing strategy
strategy based
basedon
onthe
thePb
Pb2+-DNAzyme
-DNAzyme and
and
ThT/G-quadruplex
complex.
ThT/G-quadruplex complex.
3.2. Optimization of the Detection Conditions
As a DNAzyme-based
characteristic in the system.
system.
DNAzyme-based analysis, the reaction temperature is a key characteristic
◦ C to 37 ◦ C, the signal gain
Our results
showed
that
when
we
raised
the
reaction
temperature
from
4
results showed that when we raised the reaction temperature from 4 °C to 37 °C, the signal
(F-F
increased
more than
times
0.02
to 52.17)
2), because
the because
higher temperature
gain0 )(F-F
0) increased
more2000
than
2000(from
times
(from
0.02 (Figure
to 52.17)
(Figure 2),
the higher
improved
theimproved
DNAzyme
activity,
and promoted
the promoted
reaction kinetics
of the kinetics
liquid phase
reaction
temperature
the
DNAzyme
activity, and
the reaction
of the
liquid
system.
Meanwhile,
the unspecific
DNA
was eliminated
under
temperature
phase reaction
system.
Meanwhile,
the secondary
unspecificstructure
DNA secondary
structure
washigher
eliminated
under
to
suppress
the background
noise.
higher
temperature
to suppress
the background noise.
Sensors
Sensors 2016,
2016, 16,
16, 2155
2155
Sensors 2016, 16, 2155
44 of
of 88
4 of 8
2+ sensing in the presence
result of the analysis
analysis temperature for
for Pb2+
μM
Figure
sensing in the presence of
Figure 2.
2. The
The optimization
optimization result
result of the analysis temperature
temperature forPb
Pb2+ sensing
of 11 µM
μM
2+. Original data for the optimization result of the analysis temperature shown in Figure S4.
Pb2+
.
Original
data
for
the
optimization
result
of
the
analysis
temperature
shown
in
Figure
S4.
2+
Pb . Original data for the optimization result of the analysis temperature shown in Figure S4.
Next,
in order
order to
to
verify
the
influence
of
DNAzyme
amount,
the
cleavage
reaction
was
Next,
Next, in
to verify
verify the
the influence
influence of
of DNAzyme
DNAzyme amount,
amount, the
the cleavage
cleavage reaction
reaction was
was
performed
with
different
DNAzyme
final
concentrations.
After
2
h
incubation
with
and
without
performed
performed with
with different
different DNAzyme
DNAzyme final
final concentrations.
concentrations. After 2 h incubation
incubation with and
and without
without
2+ (100 nM), the fluorescence signal gain was compared (Figure 3). As the results show, the signal
Pb
2+ (100
Pb
(100nM),
nM), the
the fluorescence
fluorescence signal
signal gain
gain was
was compared (Figure 3). As the results show,
Pb2+
show, the
the signal
signal
gain
obviously
increased
concentration
of
increased
from
0.1
to
μM,
gain
thethe
concentration
of Pb-DNAzyme
increased
from 0.1
to 0.3
because
gain obviously
obviouslyincreased
increasedasas
as
the
concentration
of Pb-DNAzyme
Pb-DNAzyme
increased
from
0.1µM,
to 0.3
0.3
μM,
because
more
DNAzyme
provided
higher
cleavage
efficiency,
generating
a
higher
signal
gain,
more
DNAzyme
provided higher
cleavage
efficiency,
generating
a higher
signalagain,
However,
because
more DNAzyme
provided
higher
cleavage
efficiency,
generating
higher
signal when
gain,
However,
when
the
concentration
of
0.3
μM,
signal
decreased
the
concentration
0.3 µM, theexceeded
signal gain
maybe
because
However,
when of
thePb-DNAzyme
concentrationexceeded
of Pb-DNAzyme
Pb-DNAzyme
exceeded
0.3decreased
μM, the
theslightly,
signal gain
gain
decreased
slightly,
because
too
background
fluorescent
signal
was
unspecific
too
muchmaybe
background
fluorescent
was produced
due to
unspecific
cleavage, due
evento
the
slightly,
maybe
because
too much
muchsignal
background
fluorescent
signal
was produced
produced
due
towithout
unspecific
2+. As a result, we chose 0.3 μM as the optimized
cleavage,
without
the
with
2+. as
combination
with
Pb2+ . As
result, we chose
0.3Pb
µM
optimized
Pb-DNAzyme
concentration
in
cleavage, even
even
without
thea combination
combination
with
Pb
Asthe
a result,
we chose
0.3 μM as
the optimized
Pb-DNAzyme
concentration
in
our
analysis.
our
analysis. concentration in our analysis.
Pb-DNAzyme
Figure 3. The optimization result of the Pb-DNAzyme concentration for Pb2+ 2+sensing in the presence of
Figure 3. The optimization result of the Pb-DNAzyme concentration for Pb 2+ sensing in the presence
Figure
optimization result
the Pb-DNAzyme
concentration
for Pb data
sensing
in optimization
the presence
2+ . [Pb-DNAzyme]
100
nM 3.
PbThe
is theofconcentration
of Pb-DNAzyme.
Original
for the
of 100 nM Pb2+
. [Pb-DNAzyme] is the concentration of Pb-DNAzyme. Original data for the
2+
2+
of 100of nM
Pb . [Pb-DNAzyme]
is theforconcentration
of Pb-DNAzyme.
result
the Pb-DNAzyme
concentration
Pb sensing shown
in Figure S5. Original data for the
optimization result of the Pb-DNAzyme concentration for Pb2+
sensing shown in Figure S5.
optimization result of the Pb-DNAzyme concentration for Pb2+ sensing shown in Figure S5.
ThT dye was
very important
for the
fluorescence signal
generation, through
recognizing and
ThT dye was
was very
very important
important for
for the
the fluorescence
fluorescence signal
signal generation,
generation, through
through recognizing
recognizing and
intercalating into the G-quadruplex structure after the G-rich
sequence
was cleaved
by DNAzyme
G-rich
sequence
cleaved
intercalating into the G-quadruplex structure after the G-rich sequence was cleaved by
by DNAzyme
DNAzyme
from the Pb-sub
strand,
although,
like
with
many
other
fluorescent
DNA
intercalators,
if too much
Pb-sub
strand,
from the Pb-sub strand, although, like with many other fluorescent DNA intercalators, if too much
dye isisadded,
a higher background
fluorescence signal
might happen
because of its unspecific
binding
dye is added,
added, aa higher
higher background
background fluorescence
fluorescence signal
signal might
might happen
happen because
because of
of its
its unspecific
unspecific
to
the double
strand
DNA
or other
DNA
second
structures.
Instructures.
order to improve
theto
signal-to-noise
binding
to
the
double
strand
DNA
or
other
DNA
second
In
order
improve
binding to the double strand DNA or other DNA second structures. In order to improve the
the
ratio,
we investigated the
relationship between
ThT concentration and ThT
the fluorescence signal,
while
signal-to-noise
signal-to-noise ratio,
ratio, we
we investigated
investigated the
the relationship
relationship between
between ThT concentration
concentration and
and the
the
all
other experiment conditions
were fixedexperiment
including the final concentration
of Pb-sub strandthe
(0.2 µM)
fluorescence
fluorescence signal,
signal, while
while all
all other
other experiment conditions
conditions were
were fixed
fixed including
including the final
final
and
Pb-DNAzyme
strand (0.3 µM).
concentration
concentration of
of Pb-sub
Pb-sub strand
strand (0.2
(0.2 μM)
μM) and
and Pb-DNAzyme
Pb-DNAzyme strand
strand (0.3
(0.3 μM).
μM).
As shown
in Figure
4, the fluorescence
signal gain increased
obviously as
the ThT concentration
As shown
shown in
in Figure
Figure 4,
4, the
the fluorescence
fluorescence signal
signal gain
gain increased
increased obviously
obviously as
as the
the ThT
ThT
increased
from
2 to 20from
µM. 2However,
when the when
concentration
of ThT of
is ThT
higher
than 20
µM,
concentration
increased
to
20
μM.
However,
the
concentration
is
higher
concentration increased from 2 to 20 μM. However, when the concentration of ThT is higher than
than 20
20
the
fluorescence
signal gain
decreased,
mainly due
to the
increase
of increase
the fluorescent
background.
μM,
the
fluorescence
signal
gain
decreased,
mainly
due
to
the
of
the
fluorescent
μM, the fluorescence signal gain decreased, mainly due to the increase of the fluorescent
Thus,
20 µM ThT was
usedThT
as an optimized
condition in all
our following
analysis.
background.
background. Thus,
Thus, 20
20 μM
μM ThT was
was used
used as
as an
an optimized
optimized condition
condition in
in all
all our
our following
following analysis.
analysis.
Sensors 2016, 16, 2155
5 of 8
Sensors 2016, 16, 2155
Sensors 2016, 16, 2155
5 of 8
5 of 8
Figure 4. The optimization result of ThT concentration for Pb2+ sensing in the presence of 100 nM
Pb2+. [ThT] is the concentration of ThT. Original data for the optimization result of ThT concentration
for Pb2+ sensing shown in Figure. S6.
3.3. Quantification of Pb2+
2+ sensing in the presence of 100 nM
2+2+
Figure
4.
Theinvestigate
optimization
result
ThTconcentration
concentration
for
Pb
In
order
the
sensitivity,
a series offorPb
solutions
prepared
with
Figure
4. to
The
optimization
result
ofofThT
Pb
sensing
inwere
the presence
of 100
nM different
Pb2+ .
2+
2+ sensor.
.
[ThT]
is
the
concentration
of
ThT.
Original
data
for
the
optimization
result
of
ThT
concentration
Pb
concentrations
and
then
analyzed
by
our
Pb
Figure
5A
shows
the
fluorescence
spectra
[ThT] is the concentration of ThT. Original data for the optimization result of ThT concentration for of
2+ sensing shown in Figure. S6.
for
Pbsensing
ThT/G-quadruplex
in the
presence
Pb2+
shown
in Figure
S6. of Pb2+ from 10 to 1000 nM. As the concentration of Pb2+
increased, the fluorescence intensity increased obviously, indicating successful DNAzyme cleavage
2+
3.3.
Quantification
Pb
of
the
Pb-Fl/Pb-Subof
3.3.
Quantification
ofduplex
Pb2+ and the formation of ThT/G-quadruplex structure.
2+a solutions
As
Figureto5Binvestigate
shows, the fluorescence
signal
gain
good linear
relationship with
with the base
In
series
of has
Pb2+
were
In order
order
to investigate the
the sensitivity,
sensitivity, aa series
of
Pb
solutions
were prepared
prepared with different
different
2+ concentration from 10 nM to 1000
2+] − 43.1832, where the [Pb2+] is
2+
10
log
of
Pb
nM:
F
=
31.6857lg[Pb
concentrations
and
then
analyzed
by
our
Pb
sensor.
Figure
5A
shows
the
fluorescence
spectra
of
2+
concentrations and then
analyzed by our Pb sensor. Figure 5A shows the fluorescence spectra
2+. The background2+signal F0 was 69.91 ± 1.23 when a blank sample was
2+
the
concentration ofinPbthe
ThT/G-quadruplex
presence
of
Pb
from
10
to
1000
nM.
As
the
concentration
of
Pb
2+
of ThT/G-quadruplex in the presence of Pb from 10 to 1000 nM. As the concentration of Pb2+
analyzed.
The limit
of detection
(LOD)
was thus
calculatedindicating
to be 0.06successful
nM, usingDNAzyme
the 3δ (3 ×cleavage
1.23) in
increased,
fluorescence
intensity
increased
obviously,
increased, the
the fluorescence
intensity
increased
obviously,
indicating successful
DNAzyme cleavage
of
the
fitting
equation.
of
Pb-Fl/Pb-Subduplex
duplex
and
the
formation
ThT/G-quadruplexstructure.
structure.
thethe
Pb-Fl/Pb-Sub
and
the
formation
ofof
ThT/G-quadruplex
As Figure 5B shows, the fluorescence signal gain has a good linear relationship with the base
10 log of Pb2+ concentration from 10 nM to 1000 nM: F = 31.6857lg[Pb2+] − 43.1832, where the [Pb2+] is
the concentration of Pb2+. The background signal F0 was 69.91 ± 1.23 when a blank sample was
analyzed. The limit of detection (LOD) was thus calculated to be 0.06 nM, using the 3δ (3 × 1.23) in
the fitting equation.
Figure 5.
5. Quantification
Quantification results
results of
of Pb
Pb2+2+: :(A)
(A)The
Thefluorescence
fluorescence spectra
spectra of
of ThT/G-quadruplex
ThT/G-quadruplexininthe
the
Figure
2+ from 10 to 1000 nM; (B) Plot for the concentration of Pb2+2+ vs. fluorescence signal gain.
2+
presence
of
Pb
presence of Pb from 10 to 1000 nM; (B) Plot for the concentration of Pb vs. fluorescence signal gain.
Errorbars
barsshow
showthe
thestandard
standarddeviations
deviationsof
ofmeasurements
measurementstaken
takenfrom
fromindependent
independentexperiments
experimentswith
with
Error
leastthree
threedistinct
distinctsensors.
sensors.
atatleast
3.4. Specificity
of 5B
Pb2+shows,
Analysis
As Figure
the
fluorescence
signal
gain has a good
linear
relationship with
Figure
5. Quantification
results
of Pb2+: (A)
The fluorescence
spectra
of ThT/G-quadruplex
in the
the base
2+
2+
2+
10 log
ofchallenge
Pb ofconcentration
10for
nM
to 1000
nM:
F = 31.6857lg[Pb
]−
43.1832,the
where
the
from specificity
10 to from
1000 nM;
(B)the
Plot
for2+the
concentration
of Pb2+investigated
vs.
fluorescence
signal
gain.[Pbof]
presence
Pb2+ the
To
Pb
sensor,
we then
response
2+
is theError
concentration
of standard
Pb .several
The
background
signal
F0 was
1.23Cu
when
a 2+blank
was
2+, Hg
2+, Cd
2+
bars show towards
the
deviations
measurements
taken
from±
independent
experiments
with
ThT/G-quadruplex
otherofdivalent
metal
ions69.91
including
, Co2+sample
, Ni
analyzed.
The
limit
of
detection
(LOD)
was
thus
calculated
to
be
0.06
nM,
using
the
3δ
(3
×
1.23)
in
the
2+ under
at least
three distinct
sensors.
and Mn
the same
analysis condition. As shown in Figure 6, our sensor generated a
fitting
equation.
significant fluorescence enhancement for 100 nM Pb2+, which is 8~100 times higher than for the
3.4. Specificity
Pb2+signal
Analysis
other
ions, andofthe
gain for some of the interfering ions was only negligible. Our results thus
3.4. Specificity of Pb2+ Analysis
2+ over other metal ions.
demonstrated
that
our
sensing
system
to Pb
To challenge the specificity
forhad
thea high
Pb2+ selectivity
sensor, we
then
investigated the response of
2+
To challenge towards
the specificity
thedivalent
Pb sensor,
we including
then investigated
response
2+
ThT/G-quadruplex
several for
other
metal ions
Cu2+, Hg2+the
, Co2+
, Ni2+, Cdof
2+ , Hg2+ , Co2+ , Ni2+ ,
ThT/G-quadruplex
towards
several
other
divalent
metal
ions
including
Cu
2+
and Mn under the same analysis condition. As shown in Figure 6, our sensor generated a
2+ under the same analysis condition. As shown in Figure 6, our sensor generated
Cd2+ and Mn
significant
fluorescence
enhancement for 100 nM Pb2+, which is 8~100 times higher than for the
a
significant
fluorescence
enhancement
forthe
100interfering
nM Pb2+ , ions
which
is only
8~100
times higher
forthus
the
other ions, and the signal gain
for some of
was
negligible.
Our than
results
other
ions,
and
the
signal
gain
for
some
of
the
interfering
ions
was
only
negligible.
Our
results
thus
2+
demonstrated that our sensing system had a high selectivity to Pb over other metal ions.
demonstrated that our sensing system had a high selectivity to Pb2+ over other metal ions.
Sensors 2016, 16, 2155
Sensors 2016, 16, 2155
6 of 8
6 of 8
2+
Figure
Figure 6.6. Investigation
Investigationresults
resultsof
ofthe
thespecificity
specificityof
ofthe
thePb
Pb2+ sensor.
sensor. The
The concentration
concentration of
of all
all the
the metal
metal
ion
solutions
was
100
nM.
All
the
analysis
experiments
were
performed
under
the
same
condition.
ion solutions was 100 nM. All the analysis experiments were performed under the same condition.
2+ sensors.
Our DNAzyme-based
DNAzyme-based sensor
sensor represents
represents aa new
new progress
progressin
inthe
thefamily
familyof
oflabel-free
label-freePb
Pb2+
Our
is an
an easy-to-operate
easy-to-operate sensor
sensor with
with 0.06
0.06 nM
nM sensitivity
sensitivity that
that exceeds
exceeds most
most of
of the
the previously
previously reported
reported
ItIt is
label-freePb
Pb2+2+ sensors
sensorsbybyseveral
several
orders
of magnitude
(Table
Wenote
alsothat
note
thisshowed
sensor
label-free
orders
of magnitude
(Table
1). We1).also
thisthat
sensor
a wider
and practical
linear
range
from
nM to 1 μM.
ashowed
wider and
practical
linear range
from
10 nM
to 10
1 µM.
Table
Table1.1.Comparison
Comparisonofofsimilar
similarPb
Pb2+2+ sensors.
sensors.
DNA Probe
DNA Probe
G-quadruplex
G-quadruplex
Linear Range
Linear Range
Detection
Detection
Methods Detection
DetectionDetection
Detection
Limit
Time
Limit
Methods
Time
1 ng/mL –1
1 ng/mL
–1 mg/mL, 1 ng/mL
1 ng/mL
mg/mL,
(4.83(4.83
nM)nM)
R2 2 = 0.992
R = 0.992
Fluorescence
Fluorescence
0.50.5
hh
Allosteric
Allosteric
G-quadruplex
G-quadruplex
DNAzyme
DNAzyme
nM–316
–316nM,
nM,
1 1nM
2 0.980
RR2 = =0.980
1 nM
1 nM
DNAzymes/
DNAzymes/
AUR
AUR
nM–1000nM,
nM,
0 0nM–1000
2 0.95
RR2 = =0.95
0.4 nM
0.4 nM
Fluorescence
Fluorescence
3.53.5
hh
G-quadruplex
G-quadruplex
DNAzyme
DNAzyme
G-quadruplex
G-quadruplex
DNAzyme
DNAzyme
5 nM–100 nM,
5 nM–100
nM,
R2 = 0.996
R2 = 0.996
10 nM–1000 nM,
10 nM–1000
nM,
R2 = 0.997
R2 = 0.997
3 nM
3 nM
Fluorescence
Fluorescence
3.5 h
3.5 h
0.06 nM
0.06 nM
Fluorescence
Fluorescence
2.5 h
2.5 h
Chemiluminescence 2 h
Chemiluminescence
2h
Reaction
Reference
Reaction
Reference
System
System
DsDNA’s
DsDNA’s
conformational
[25]
conformational
[25]
changes/
changes/PicoGreen
PicoGreen
luminol-H
2 O22O2
luminol-H
[26]
[26]
G4/hemin/H
G4/hemin/H22O
O22//
AUR
catalytic
[27]
AUR
catalytic
[27]
system
system
catalytic
catalytic
[28]
beacons/G4/ZnPPIX
beacons/G4/
[28]
ZnPPIX
G4/ThT
This work
G4/ThT
This work
4. Conclusions
4. Conclusions
In this work, we constructed a novel Pb2+
fluorescence sensor, based on a DNAzyme directed
In this work, we constructed a novel Pb2+ fluorescence sensor, based on a DNAzyme directed
G-quadruplex/ThT conformation. Our results demonstrated the excellent sensing performance of
G-quadruplex/ThT conformation. Our results demonstrated the excellent sensing performance of
the DNAzyme when Pb2+
was added. The sensitivity was 0.06 nM which is much lower than
the DNAzyme when Pb2+ was added. The sensitivity
was 0.06 nM which is much lower than
EPA-defined maximal contamination level for Pb2+ in drinking
water (72 nM) and is, to our knowledge,
2+
EPA-defined maximal contamination level for Pb in drinking water (72 nM) and is, to our
quite competitive among all the reported label-free Pb2+ sensors. The
analysis was conveniently
knowledge, quite competitive among all the reported label-free Pb2+ sensors. The analysis was
performed in one tube in two simple steps, avoiding unnecessary contamination and oxidation,
conveniently performed in one tube in two simple steps, avoiding unnecessary contamination and
thus our quantification results were so stable that the relative standard deviations (RSD) were all
oxidation, thus our quantification results were so stable that the relative standard deviations (RSD)
below 5% except for only the RSD for 10 nM Pb2+ (11%). Importantly,
good selectivity was achieved
were all below 5% except for only the RSD for 10 nM Pb2+ (11%). Importantly, good selectivity was
compared with six different metal ions, indicating a great potential application of our sensor for assays
achieved compared with six different metal ions, indicating a great potential application of our
in complex samples.
sensor for assays in complex samples.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/16/12/2155/
Supplementary
Materials:
The
following
are
available
online
at
s1.
Figure S1. CD spectrum
of different samples.
Figure
S2. A time-dependent
amplification
of the
fluorescence
http://www.mdpi.com/1424-8220/16/12/2155/s1.
Figure S1. CD spectrum
of different
Figure
S2. A
sensor
for 50 nM Pb2+ detection. Figure S3. Polyacrylamidegel
electrophoresis
resultssamples.
of different
samples.
◦ C; (B) 25 ◦ C; (C) 37 ◦ C in the
Figure
S4. Original
data for the optimization
result of
the analysis
temperature
(A) 4 Figure
S3. Polyacrylamidegel
time-dependent
amplification
of the fluorescence
sensor
for 50 nM
Pb2+ detection.
2+
2+
presence
of 1 µMresults
Pb . Figure
S5. Original
data
of 100S4.
nM
Pb sensing
in the
of different
concentration
electrophoresis
of different
samples.
Figure
Original
data for
the presence
optimization
result of
the analysis
2+ sensing
of
Pb-DNAzyme:
(B) 0.2
(C)
(D) 0.5ofµM.
Figure
Original
data of 100
temperature
(A) 4(A)
°C;0.1
(B)µM;
25 °C;
(C) µM;
37 °C
in0.3
theµM;
presence
1 μM
Pb2+.S6.
Figure
S5. Original
datanM
of Pb
100 nM
Pb2+
using different concentration of ThT: (A) 2 µM; (B) 5 µM; (C) 10 µM; (D) 20 µM; (E) 30 µM; (F) 50 µM.
sensing in the presence of different concentration of Pb-DNAzyme: (A) 0.1 μM; (B) 0.2 μM; (C) 0.3 μM; (D) 0.5
μM. Figure S6. Original data of 100 nM Pb2+ sensing using different concentration of ThT: (A) 2 μM; (B) 5 μM;
(C) 10 μM; (D) 20 μM; (E) 30 μM; (F) 50 μM.
Sensors 2016, 16, 2155
7 of 8
Acknowledgments: The authors would like to thank the financial support from the National Natural Science
Foundation of China (No. 21305091) and the General Administration of Quality Supervision, Inspection and
Quarantine of the People’s Republic of China (2014QK141).
Author Contributions: G.L., Y.W. and L.W. designed the experiments and wrote the main manuscript text, Y.W.,
L.W.; L.L. and L.X. performed the experiments. All authors reviewed the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Raj, S.; Shankaran, D.R. Curcumin based biocompatible nanofibers for lead ion detection. Sens. Actuators
B Chem. 2016, 226, 318–325. [CrossRef]
Shang, Y.; Zhang, Y.; Li, P.; Lai, J.; Kong, X.-Y.; Liu, W.; Xiao, K.; Xie, G.; Tian, Y.; Wen, L. DNAzyme tunable
lead (II) gating based on ion-track etched conical nanochannels. Chem. Commun. 2015, 51, 5979–5981.
[CrossRef] [PubMed]
Liu, J.; Lu, Y. A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. J. Am.
Chem. Soc. 2003, 125, 6642–6643. [CrossRef] [PubMed]
Kwon, J.Y.; Jang, Y.J.; Lee, Y.J.; Kim, K.M.; Seo, M.S.; Nam, W.; Yoon, J. A highly selective fluorescent
chemosensor for Pb2+ . J. Am. Chem. Soc. 2005, 127, 10107–10111. [CrossRef] [PubMed]
Wen, Y.Q.; Peng, C.; Li, D.; Zhuo, L.; He, S.J.; Wang, L.H.; Huang, Q.; Xu, Q.H.; Fan, C.H. Metal ion-modulated
graphene-DNAzyme interactions: Design of a nanoprobe for fluorescent detection of lead(II) ions with high
sensitivity, selectivity and tunable dynamic range. Chem. Commun. 2011, 47, 6278–6280. [CrossRef] [PubMed]
Behbahani, M.; Hassanlou, P.G.; Amini, M.M.; Omidi, F.; Esrafili, A.; Farzadkia, M.; Bagheri, A. Application
of solvent-assisted dispersive solid phase extraction as a new, fast, simple and reliable preconcentration and
trace detection of lead and cadmium ions in fruit and water samples. Food Chem. 2015, 187, 82–88. [CrossRef]
[PubMed]
Barbosa, V.M.P.; Barbosa, A.F.; Bettini, J.; Luccas, P.O.; Figueiredo, E.C. Direct extraction of lead (II) from
untreated human blood serum using restricted access carbon nanotubes and its determination by atomic
absorption spectrometry. Talanta 2016, 147, 478–484. [CrossRef] [PubMed]
Ma, R.; Van Mol, W.; Adams, F. Determination of cadmium, copper and lead in environmental samples.
An evaluation of flow injection on-line sorbent extraction for flame atomic absorption spectrometry.
Anal. Chim. Acta 1994, 285, 33–43. [CrossRef]
Jackson, S.E.; Pearson, N.J.; Griffin, W.L.; Belousova, E.A. The application of laser ablation-inductively
coupled plasma-mass spectrometry to in situ U-Pb zircon geochronology. Chem. Geol. 2004, 211, 47–69.
[CrossRef]
Liang, Q.; Jing, H.; Gregoire, D.C. Determination of trace elements in granites by inductively coupled plasma
mass spectrometry. Talanta 2000, 51, 507–513. [CrossRef]
Elfering, H.; Andersson, J.T.; Poll, K.G. Determination of organic lead in soils and waters by hydride
generation inductively coupled plasma atomic emission spectrometry. Analyst 1998, 123, 669–674. [CrossRef]
Ochsenkühn-Petropoulou, M.; Ochsenkühn, K.M. Comparison of inductively coupled plasma-atomic
emission spectrometry, anodic stripping voltammetry and instrumental neutron-activation analysis for
the determination of heavy metals in airborne particulate matter. Fresenius’ J. Anal. Chem. 2001, 369, 629–632.
[CrossRef]
Liu, J.; Cao, Z.; Lu, Y. Functional nucleic acid sensors. Chem. Rev. 2009, 109, 1948–1998. [CrossRef] [PubMed]
Liu, J.; Lu, Y. A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in aqueous solution with
high sensitivity and selectivity. J. Am. Chem. Soc. 2007, 129, 9838–9839. [CrossRef] [PubMed]
Liu, J.; Brown, A.K.; Meng, X.; Cropek, D.M.; Istok, J.D.; Watson, D.B.; Lu, Y. A catalytic beacon sensor for
uranium with parts-per-trillion sensitivity and millionfold selectivity. Proc. Natl. Acad. Sci. USA 2007, 104,
2056–2061. [CrossRef] [PubMed]
Hollenstein, M.; Hipolito, C.; Lam, C.; Dietrich, D.; Perrin, D.M. A highly selective DNAzyme sensor for
mercuric ions. Angew. Chem. Int. Ed. 2008, 47, 4346–4350. [CrossRef] [PubMed]
Lan, T.; Furuya, K.; Lu, Y. A highly selective lead sensor based on a classic lead DNAzyme. Chem. Commun.
2010, 46, 3896–3898. [CrossRef] [PubMed]
Sensors 2016, 16, 2155
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
8 of 8
Zhao, L.Y.; Gu, W.; Zhang, C.L.; Shi, X.H.; Xian, Y.Z. In situ regulation nanoarchitecture of Au
nanoparticles/reduced graphene oxide colloid for sensitive and selective SERS detection of lead ions.
J. Colloid Interface Sci. 2016, 465, 279–285. [CrossRef] [PubMed]
Zhang, X.-B.; Wang, Z.; Xing, H.; Xiang, Y.; Lu, Y. Catalytic and Molecular Beacons for Amplified Detection
of Metal Ions and Organic Molecules with High Sensitivity. Anal. Chem. 2010, 82, 5005–5011. [CrossRef]
[PubMed]
Kim, J.H.; Han, S.H.; Chung, B.H. Improving Pb2+ detection using DNAzyme-based fluorescence sensors by
pairing fluorescence donors with gold nanoparticles. Biosens. Bioelectron. 2011, 26, 2125–2129. [CrossRef]
[PubMed]
Xiao, Y.; Rowe, A.A.; Plaxco, K.W. Electrochemical detection of parts-per-billion lead via an electrode-bound
DNAzyme assembly. J. Am. Chem. Soc. 2007, 129, 262–263. [CrossRef] [PubMed]
Dalavoy, T.S.; Wernette, D.P.; Gong, M.; Sweedler, J.V.; Lu, Y.; Flachsbart, B.R.; Shannon, M.A.; Bohn, P.W.;
Cropek, D.M. Immobilization of DNAzyme catalytic beacons on PMMA for Pb 2+ detection. Lab Chip 2008, 8,
786–793. [CrossRef] [PubMed]
Chiuman, W.; Li, Y. Efficient signaling platforms built from a small catalytic DNA and doubly labeled
fluorogenic substrates. Nucleic Acids Res. 2007, 35, 401–405. [CrossRef] [PubMed]
Du, Y.; Li, B.; Wang, E. “Fitting” Makes “Sensing” Simple: Label-Free Detection Strategies Based on Nucleic
Acid Aptamers. Accounts Chem. Res. 2013, 46, 203–213. [CrossRef] [PubMed]
Huang, Y.; Yan, J.; Fang, Z.; Zhang, C.; Bai, W.; Yan, M.; Zhu, C.; Gao, C.; Chen, A. Highly sensitive and
selective optical detection of lead(II) using a label-free fluorescent aptasensor. RSC Adv. 2016, 6, 90300–90304.
[CrossRef]
Li, T.; Wang, E.; Dong, S. Lead(II)-induced allosteric G-quadruplex DNAzyme as a colorimetric and
chemiluminescence sensor for highly sensitive and selective Pb2+ detection. Anal. Chem. 2010, 82, 1515–1520.
[CrossRef] [PubMed]
Li, C.; Liu, K.; Lin, Y.; Chang, H. Fluorescence detection of Lead(II) ions through their induced catalytic
activity of DNAzymes. Anal. Chem. 2011, 83, 225–230. [CrossRef] [PubMed]
Fu, T.; Ren, S.L.; Gong, L.; Meng, H.M.; Cui, L.; Kong, R.M.; Zhang, X.B.; Tan, W.H. A label-free DNAzyme
fluorescence biosensor for amplified detection of Pb2+ -based on cleavage-induced G-quadruplex formation.
Talanta 2016, 147, 302–306. [CrossRef] [PubMed]
Chen, J.; Lin, J.; Zhang, X.; Cai, S.; Wu, D.; Li, C.; Yang, S.; Zhang, J. Label-free fluorescent biosensor based
on the target recycling and Thioflavin T-induced quadruplex formation for short DNA species of c-erbB-2
detection. Anal. Chim. Acta 2014, 817, 42–47. [CrossRef] [PubMed]
Mohanty, J.; Barooah, N.; Dhamodharan, V.; Harikrishna, S.; Pradeepkumar, P.I.; Bhasikuttan, A.C. Thioflavin
T as an Efficient Inducer and Selective Fluorescent Sensor for the Human Telomeric G-Quadruplex DNA.
J. Am. Chem. Soc. 2013, 135, 367–376. [CrossRef] [PubMed]
Gabelica, V.R.; Maeda, R.; Fujimoto, T.; Yaku, H.; Murashima, T.; Sugimoto, N.; Miyoshi, D. Multiple and
cooperative binding of fluorescence light-up probe thioflavin t with human telomere DNA G-Quadruplex.
Biochemistry 2013, 52, 5620–5628. [CrossRef] [PubMed]
Faverie, A.R.; Guédin, A.; Bedrat, A.; Yatsunyk, L.A.; Mergny, J.-L. Thioflavin T as a fluorescence light-up
probe for G4 formation. Nucleic Acids Res. 2014. [CrossRef]
© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).