sensors
Article
Label-Free Fluorescence Assay of S1 Nuclease and
Hydroxyl Radicals Based on Water-Soluble
Conjugated Polymers and WS2 Nanosheets
Junting Li, Qi Zhao and Yanli Tang *
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key Laboratory of Applied
Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering,
Shaanxi Normal University, Xi’an 710062, China; [email protected] (J.L.); [email protected] (Q.Z.)
* Correspondence: [email protected]; Tel.: +86-29-8153-0844
Academic Editors: Alexandre François, Al Meldrum and Nicolas Riesen
Received: 15 April 2016; Accepted: 8 June 2016; Published: 13 June 2016
Abstract: We developed a new method for detecting S1 nucleaseand hydroxyl radicals based on the
use of water-soluble conjugated poly[9,9-bis(6,6-(N,N,N-trimethylammonium)-fluorene)-2,7-ylenevinylene
-co-alt-2,5-dicyano-1,4-phenylene)] (PFVCN) and tungsten disulfide (WS2 ) nanosheets. Cationic
PFVCN is used as a signal reporter, and single-layer WS2 is used as a quencher with a negatively
charged surface. The ssDNA forms complexes with PFVCN due to much stronger electrostatic
interactions between cationic PFVCN and anionic ssDNA, whereas PFVCN emits yellow fluorescence.
When ssDNA is hydrolyzed by S1 nuclease or hydroxyl radicals into small fragments, the interactions
between the fragmented DNA and PFVCN become weaker, resulting in PFVCN being adsorbed
on the surface of WS2 and the fluorescence being quenched through fluorescence resonance energy
transfer. The new method based on PFVCN and WS2 can sense S1 nuclease with a low detection
limit of 5 ˆ 10´6 U/mL. Additionally, this method is cost-effective by using affordable WS2 as an
energy acceptor without the need for dye-labeled ssDNA. Furthermore, the method provides a new
platform for the nuclease assay and reactive oxygen species, and provides promising applications for
drug screening.
Keywords: S1 nuclease; hydroxyl radicals; fluorescent sensing; label-free; conjugated polymers;
WS2 nanosheet
1. Introduction
The detection of endonuclease activity is essential because endonuclease plays an important role
in many biological processes. Endonuclease has been widely used as a tool to remove non-annealed
polynucleotide tails and hairpin loops in RNA and DNA duplexes, molecular cloning, and gene
analysis [1–3]. S1 nuclease, a well-known multifunctional endonuclease, can hydrolyze ssDNA or RNA
into 5’-phosphomononucleotide and 5’-phosphooligonucleotide and is employed as a model system.
S1 nuclease has been particularly used to probe the disruption of the DNA structure by numerous
carcinogens and antimitotic drugs [4]. Numerous methods based on advanced materials have been
developed to detect S1 nuclease, such as colorimetric, electrochemical, and fluorescent assays [5–10].
However, most of these methods have some drawbacks, such as low detection limits, high cost and/or
time-consuming assay procedures.
Hydroxyl radicals are highly reactive in many biological processes and can damage
virtually all types of macromolecules, including carbohydrates, nucleic acids, lipids, and amino
acids [11–14]. Hydroxyl radicals have received increasing attention due to their function in
mutagenesis, carcinogenesis, and aging [15–18]. The DNA damage caused by hydroxyl radicals
Sensors 2016, 16, 865; doi:10.3390/s16060865
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generates characteristic mutagenic base lesions and strand fragments in cellular systems [15,19].
To monitor DNA damage caused by hydroxyl radicals, methods based on the FRET technique were
established [7,9,20–22]. Nevertheless, considering the crucial roles of S1 nuclease and hydroxyl radicals
in many biological events, it is important to develop simple and convenient strategies for their analysis.
Recently, single- and few-layered two-dimensional (2D) transition metal dichalcogenides
(TMDCs), as planar covalent-network solids, have attracted growing attention in the fields of
electronics, sensors, optics, catalysis, and energy harvesting, due to their special structures with high
specific surface area and remarkable electronic properties [23–25]. Layered tungsten disulfide (WS2 ),
one of the newly emerging 2D TMDCs, which consists of S´W´S sandwiches in a trigonal prismatic
coordination, has been recognized as a novel nanomaterial in biomedical applications [26–28] because
the large lateral dimensions and high surface areas of layered WS2 can effectively quench tagged
fluorophores [29]. However, few bioassays based on WS2 nanosheets have been developed [29–33].
Thus, it is of interest to explore their new application as a biosensing platform.
Various conjugated polymer materials, especially water-soluble conjugated polymers (WSCPs),
have been studied widely as useful platforms for sensitive biosensors and chemosensors [34–37].
WSCPs are composed of a large number of conjugated repeated units, which provides strong absorption
and emission in the Ultraviolet´Visible light (UV–vis) range. When WSCPs are excited, the excitation
energy along the main chain can rapidly transfer to an energy or electron acceptor, which accounts for
the fluorescence signal amplification [35,36]. Thus, sensors based on WSCPs provide sensitive and
specific platforms for various targets, such as oligonucleotides, enzymes, proteins, inhibitors, and toxic
metal ions [7,37–44]. However, to the best of our knowledge, WSCPs and WS2 nanosheets used as a
sensing platform have not been reported.
In this paper, we design a new approach to detect the activity of the S1 nuclease and hydroxyl
radicals based on water-soluble PFVCN and WS2 nanosheets. In our strategy, PFVCN can be absorbed
on the surface of single-layer WS2 nanosheets via electrostatic interactions, and WS2 nanosheets act as
an efficient quencher for PFVCN [29]. When ssDNA is added, the fluorescence of PFCVN is recovered
to some extent because of the much stronger electrostatic interaction between PFVCN and the ssDNA
probe, leading to PFVCN leaving the surface of WS2 . Upon addition of S1 nuclease or hydroxyl radicals,
the ssDNA probe is hydrolyzed into small fragments, and PFVCN is adsorbed on the nanosheets,
resulting in the fluorescence of PFVCN being quenched. Thus, S1 nuclease activity and hydroxyl
radicals can be measured by monitoring the PFVCN fluorescence intensity, which is cost-effective,
sensitive and label-free without the need for dye labeling of the ssDNA. This platform composed of
WSCPs and WS2 also provides a new sensing system for biosensors.
2. Materials and Methods
2.1. Materials and Measurements
The oligonucleotides, ATP and BSA were purchased from Shanghai Sangon Biological
Engineering Technology & Service Co. Ltd. (Shanghai, China). S1 nuclease and thiourea
were obtained from Sigma (St. Louis, MO, USA). EcoRI, Exonuclease III and Klenow fragment
polymerase (KF polymerase) were purchased from Dalian Takara Biotechnology Co. Ltd. (Dalian,
China). The monolayer tungsten disulfide (WS2 ) nanosheet was purchased from Nanjing XFNano
Material Tech. Co. Ltd. (Nanjing, China) Cationic poly[9,9-bis(6,6-(N,N,N-trimethylaminium)fluorene)-2,7-ylenevinylene-co-alt-2,5-dicyano-1,4-phenylene] (PFVCN) was synthesized according to
the literature [45]. The oligonucleotide sequence used in our experiments was as follows: 51 -CAA TGG
AAC TAT TCG GCA TCA ATA CTC ATC-31 . The concentration of oligonucleotide was determined by
measuring the absorbance at 260 nm in a 250 µL quartz cuvette. UV´vis absorption spectra were taken
on a Lambda 35 spectrophotometer (Perkin Elmer, Waltham , MA, USA). The fluorescence spectra
were recorded on a F-7000 spectrophotometer (Hitachi, Hitachi, Tokyo, Japan) equipped with a xenon
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lamp excitation source. The zeta potentials were measured on a Nano-ZS90 (Malvern Instruments,
Malvern, UK). All solutions were prepared with ultrapure water from a Millipore filtration system.
2.2. Procedure
2.2.1. Enzyme Assay
Forty microliters of the mixture containing 10 nM ssDNA probe and various concentrations of
S1 nuclease was incubated in an enzyme reaction buffer (60 mM CH3 COONa, 200 mM NaCl, and
2 mM Zn(SO4 )2 , pH 4.6) at 37 ˝ C for 0.5 h. The mixture was heated at 95 ˝ C for 10 min to terminate
the cleavage reaction. Then, the reaction solution was diluted with Tris-HCl buffer (20 mM, pH 7.4)
before the addition of WS2 nanosheets (1 µg/mL). After incubating the mixture for 10 min at 25 ˝ C,
PFVCN solution (10´6 M in repeat units) was added to the reaction with a final volume of 2 mL. The
final concentration of S1 nuclease ranged from 5.0 ˆ 10´6 U/mL to 0.7 U/mL. The emission spectra
were measured at an excitation wavelength of 470 nm at 25 ˝ C.
2.2.2. Assay for S1 Nuclease as a Function of Incubating Time
A series of reaction mixtures containing a fixed concentration of ssDNA probe and S1 nuclease
were incubated at 37 ˝ C for 0, 5, 10, 15, 20, 25, 30, and 35 min, respectively. The subsequent experimental
procedure was the same as the enzyme assay.
2.2.3. Inhibition Assay by ATP
For enzyme inhibition experiments, a mixture containing 0.5 U/mL S1 nuclease and various
concentrations of ATP ranging from 0 to 70 µM was incubated before the addition of the ssDNA probe.
The subsequent procedure was the same as the enzyme assay.
2.2.4. Specificity Assay of S1 Nuclease
To verify the specificity of S1 nuclease, ExoIII, EcoRI, BSA and KF polymerase were used to replace
S1 nuclease. According to the aforementioned experimental procedure, the fluorescence spectra were
measured under the same conditions.
2.2.5. ¨ OH assay and Inhibition Assay by Thiourea
In 2.0 mL 20 mM Tris-HCl buffer (pH 7.0), the solution of the ssDNA probe (10 nM) was treated
with various concentrations of Fenton’s agent ([Fe2+ ]:[H2 O2 ]:[DTT] = 1:10:10) for 5 min before the
addition of WS2 nanosheets (1 µg/mL). After incubating the mixture for 10 min at 25 ˝ C, the PFVCN
solution (10´6 M) was added. The concentration of hydroxyl radicals was determined according to
Fe2+ , which ranged from 0.01 µM to 5 µM. The emission spectra were measured with the excitation
wavelength of 470 nm at 25 ˝ C. The inhibition experiments were the same as in the above procedure,
except for the involvement of thiourea in the reaction solution before the addition of the ssDNA probe.
3. Results and Discussion
3.1. Array Mechanism
The proposed principle of the detection of S1 nuclease and hydroxyl radicals is illustrated in
Scheme 1. Water-soluble cationic PFVCN is used as the optical transducer in the biosensor, and WS2 is
used as a fluorescence quencher. WS2 nanosheets were purchased commercially with the thickness
of ~1.0 nm and the size in the range of 20–500 nm, which was provided by the supplier. Also, we
obtained the TEM image of WS2 that is coincident with data from the supplier (Figure S1). The PFVCN
can be absorbed on the surface of single-layer WS2 nanosheets via electrostatic interactions. According
to the information provided by the supplier, the WS2 was prepared through a controllable lithiation
process leading to the WS2 having a negative charge [46] (the ζ potential of WS2 was measured
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as ´26.0 ˘ 0.6 mV), resulting in substantial fluorescence quenching through fluorescence resonance
energy transfer [32,47–49]. Upon the addition of ssDNA, PFVCN can form complexes with ssDNA
due to the stronger electrostatic interaction between them. PFVCN then leaves the surface of the WS2
nanosheets and emits an enhanced fluorescence signal. When ssDNA is cleaved by S1 nuclease or
¨ OH into small fragments, the PFVCN/ssDNA complexes do not form because of their weaker affinity,
Sensors
2016, 16, 865on the nanosheets, causing the fluorescence of PFVCN to be quenched.
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and PFVCN is
adsorbed
Figure
S2a shows that
the
PFVCN
fluorescence
change
is
coincident
with
the
proposed
principle.
Control
nuclease or .OH into small fragments, the PFVCN/ssDNA complexes do not form because of their
experimentsweaker
showaffinity,
that S1
and hydroxyl
radicals
have
no obvious
effecttoon
andnuclease
PFVCN is adsorbed
on the nanosheets,
causing
the fluorescence
of PFVCN
be the PFVCN
shows that
PFVCN of
fluorescence
changeS2b,d).
is coincident
with
proposed
fluorescence quenched.
intensityFigure
in theS2a
presence
orthe
absence
WS2 (Figure
Also,
thethefluorescence
intensity
principle. Control experiments show that S1 nuclease and hydroxyl radicals have no obvious effect
of PFVCN keeps
stable
in
the
presence
of
ssDNA
without
WS
(Figure
S2c).
Consequently,
the
changes
2 of WS2 (Figure S2b,d). Also, the
on the PFVCN fluorescence intensity in the presence or absence
of the fluorescence
intensity
PFVCN
canstable
be used
to detect
S1 nuclease
and
OH and
fluorescence
intensity of
of PFVCN
keeps
in the presence
of ssDNA
without WS
2 (Figure
S2c). to sense the
the changes
the fluorescence
intensity
PFVCN can be used to detect S1 nuclease
hydrolysis ofConsequently,
ssDNA without
theofneed
for a label
on theof ssDNA.
and OH and to sense the hydrolysis of ssDNA without the need for a label on the ssDNA.
Scheme 1. Proposed Strategy for the Hydrolysis of ssDNA by S1 Nuclease or Hydroxyl Radicals and
the Chemical
Structurefor
of PFVCN
Scheme 1. Proposed
Strategy
the Hydrolysis of ssDNA by S1 Nuclease or Hydroxyl Radicals and
the Chemical Structure of PFVCN
3.2. Optimization of the Experimental Conditions
To study the effect of the concentration and length of the nucleotide acids on the degree of
3.2. Optimization
of the recovery,
Experimental
Conditions
fluorescence
we investigated
the fluorescence intensity of PFVCN (1.0 × 10−6 M in RUs) in
the presence of the ssDNA probe in different concentrations. As shown in Figure S3a, WS2 nanosheets
To study
the effect of the concentration and length of the nucleotide acids on the degree of
have a maximum absorption at 258 nm and emit very weak fluorescence with the excitation
´6
fluorescence wavelength
recovery,atwe
the fluorescence
intensity
PFVCN
ˆ×10
258investigated
nm. The concentration
of WS2 was optimized
first of
in the
presence(1.0
of 1.0
10−6 MM in RUs) in
PFVCN.
When
the
concentration
of
WS
2 is 1 μg/mL, the fluorescence intensity of PFVCN is quenched
the presence of the ssDNA probe in different concentrations. As shown in Figure S3a, WS2 nanosheets
greatly and reaches the plateau (Figure S3b). Thus, 1.0 × 10−6 M PFVCN and 1 μg/mL WS2 were used
have a maximum
absorption at 258 nm and emit very weak fluorescence with the excitation wavelength
in following experiments. Furthermore, the control experiments also showed that the fluorescence of
at 258 nm. The
concentration
first
in the
presence
1.0ofˆssDNA
10´6 M PFVCN.
PFVCN/WS
2 system wasof
notWS
interfered
S1 and hydroxyl
radicals
(Figure
S2c). Theof
effect
2 wasbyoptimized
concentration
on
PFVCN/WS
2 system fluorescence was shown in Figure 1a. The fluorescence
When the concentration of WS2 is 1 µg/mL, the fluorescence intensity of PFVCN is quenched greatly
intensity gradually increased with the increase of the ssDNA
probe concentration, resulting from the
and reaches increased
the plateau
(Figure S3b). Thus, 1.0 ˆ 10´6 M PFVCN and 1 µg/mL WS2 were used in
number of PFVCN complexes with ssDNA leaving the surface of WS2. When the
following experiments.
the control
showed
thatreached
the fluorescence
of
concentration ofFurthermore,
ssDNA was increased
to 10 nM,experiments
the fluorescencealso
intensity
of PFVCN
a
To was
investigate
the effect of the
acid base length
on the fluorescence
enhancement,
PFVCN/WS2plateau.
system
not interfered
bynucleotide
S1 and hydroxyl
radicals
(Figure S2c).
The effect of ssDNA
ssDNA (10 nM) with different base lengths, varying from 7- to 30-mer, were tested. As shown in
concentrationFigure
on PFVCN/WS
fluorescence was shown in Figure 1a. The fluorescence intensity
2 system
1b, the fluorescence
intensity of PFVCN gradually increased with the increase of the ssDNA
gradually increased
increase
of the
ssDNAbetween
probePFVCN
concentration,
resulting
from
length duewith
to the the
stronger
electrostatic
interaction
and the longer
ssDNA. When
thethe increased
length of ssDNA
was a 30-mer,
fluorescence
of PFVCN
reached a maximum;
the rate
of
number of PFVCN
complexes
with the
ssDNA
leaving
the surface
of WS2 . however,
When the
concentration
of
fluorescence increase became slower. These results indicate that the fluorescence enhancement of
ssDNA was increased
to 10 nM, the fluorescence intensity of PFVCN reached a plateau. To investigate
PFVCN is dependent on concentration and length of the ssDNA. Accordingly, 30-mer ssDNA at a
the effect of the
nucleotide
acid
base
length
the fluorescence
enhancement,
ssDNA (10 nM) with
concentration
of 10 nM
was
chosen
for theon
following
analytical studies.
This method is cost-effective
to use ofvarying
cheap WS2from
as an energy
acceptor without
the need for
ssDNA.
different basedue
lengths,
7- to 30-mer,
were tested.
Asdye-labelling
shown in the
Figure
1b, the fluorescence
intensity of PFVCN gradually increased with the increase of the ssDNA length due to the stronger
electrostatic interaction between PFVCN and the longer ssDNA. When the length of ssDNA was
a 30-mer, the fluorescence of PFVCN reached a maximum; however, the rate of fluorescence increase
became slower. These results indicate that the fluorescence enhancement of PFVCN is dependent on
concentration and length of the ssDNA. Accordingly, 30-mer ssDNA at a concentration of 10 nM was
Sensors 2016, 16, 865
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chosen for the following analytical studies. This method is cost-effective due to use of cheap WS2 as an
energy acceptor without the need for dye-labelling the ssDNA.
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Figure 1. (a) Fluorescence intensity of PFVCN/ssDNA in Tris-HCl buffer solution (20 mM, pH 7.4)
with different intensity
concentrations.of
[ssDNA]
= 0−12 nM; (b) Fluorescence
intensitybuffer
of PFVCN/ssDNA
Figure 1. (a) Fluorescence
PFVCN/ssDNA
in Tris-HCl
solutionin (20 mM, pH 7.4)
Tris-HCl buffer solution (20 mM, pH 7.4) with different base lengths. [PFVCN] = 1.0 × 10−6 M,
with different concentrations.
[ssDNA]
=
0´12
nM;
(b)
Fluorescence
intensity
of PFVCN/ssDNA
[ssDNA] = 10 nM, [WS2] = 1 μg/mL. The error bars represent the standard deviations of three parallel
wavelength
is 470 nm.
in Tris-HCl buffermeasurements.
solution The
(20excitation
mM, pH
7.4) with
different base lengths. [PFVCN] = 1.0 ˆ 10´6 M,
[ssDNA] = 10 3.3.
nM,
[WS2 ]S1=Nuclease
1 µg/mL. The error bars represent the standard deviations of three parallel
Sensing of
measurements. The
excitation
wavelength is 470 nm.
We then investigated the detection of S1 by measuring the fluorescence spectra under various
concentrations of S1. The final concentration in the samples ranged from 0 to 0.7 U/mL. As shown in
Figure 2a, the emission of PFVCN decreases with the increasing concentration of S1 from 0 to
3.3. Sensing of S1 Nuclease
0.5 U/mL. The ssDNA substrate was cleaved into small fragments by S1, which reduced the
Figure 1. (a) Fluorescence intensity of PFVCN/ssDNA in Tris-HCl buffer solution (20 mM, pH 7.4)
interactions between PFVCN and ssDNA and prompted the adsorption of PFVCN on the WS2 surface.
We then investigated
the
detection
S1
by
measuring
fluorescence
spectra
with
different
[ssDNA]
= 0−12
nM;
(b)1b.Fluorescence
intensity
ofthe
PFVCN/ssDNA
These
resultsconcentrations.
are
consistent
with
theof
results
in Figure
Figure 2bthe
demonstrates
relationship
ofin under various
−6 M,
Tris-HCl
buffer
solution
(20
mM,
pH
7.4)
with
different
base
lengths.
[PFVCN]
=
1.0
×
10
the
fluorescence
intensity
ratio
(I
/I)
of
PFVCN
to
the
concentration
of
S1,
where
I
and
I
are
the
concentrations of S1. The final concentration in the samples ranged from 0 to 0.7 U/mL. As shown in
[ssDNA]
= 10 nM,
[WS2] =of1 sensor
μg/mL.solution
The error
represent
standard
three parallel
fluorescence
intensities
in bars
the absence
andthe
presence
of deviations
S1 nuclease,ofrespectively.
Figure 2a, the emission
PFVCN
concentration
S1 from 0 to 0.5 U/mL.
The ratioof
increases
withdecreases
increasing
of with
S1 isconcentration,
which means
the fluorescence of
of PFVCN
measurements.
The excitation
wavelength
470the
nm.increasing
decreases
to quenching
WS fragments
upon ssDNA analysis
S1. The
detection the
limit interactions
was
The ssDNA substrate
wasdue
cleaved
into by
small
by S1, by
which
reduced
between
5 × 10 ofU/mL
(the signal at the detection limit (S ) is given by: S = S − 3 * σ , where S is the signal
3.3. Sensing
S1 Nuclease
PFVCN and ssDNA
andwithout
prompted
the
adsorption
of PFVCN
onsignal
the from
WS11
2 surface.
for a blank
S1, σ is the
known
standard deviation
for the blank’s
experiments.These results are
investigated
of and
S1 by
thewhich
fluorescence
spectra
In then
this
case,
S in
, S Figure
, andthe
σ detection
are
275,
281,
1.5,measuring
respectively.),
is lower
than
that under
reported
consistent with We
the
results
1b.
Figure
2b
demonstrates
the
relationship
ofvarious
the fluorescence
concentrations
S1. The
final
the samples
ranged
0 to× 0.7
shown in
previouslyof by
Zhang
et concentration
al. ( 5 × 10 inU/mL)
[50], Yuan
et from
al. (1.4
10 U/mL.
U/μL)As[51],
intensity ratio
(I
/I)
of
PFVCN
to
the
concentration
of
S1,
where
I
and
I
are
the
fluorescence
0 Zhou
0 concentration
et al.emission
(0.04 U/mL)of[10]
and Chudecreases
et al. (5 × 10with
U/μL)
[52].
He et al. studied
the interaction
Figure
2a, the
PFVCN
the
increasing
of between
S1 from 0 to intensities
ssDNA
and
graphene
oxide
and
developed
a
GO
based
biosensor
for
S1
nuclease
with
the
detection
of sensor solution
in The
the ssDNA
absence
and presence
of into
S1 nuclease,
respectively.
The
ratio increases
with
0.5 U/mL.
substrate
was cleaved
small fragments
by S1, which
reduced
the
limit of 5.8 × 10 U/mL [53], which is also higher than that obtained in this work. When the S1
between PFVCN
and ssDNA
and
prompted
the adsorption
of PFVCN
on the WS2 due
surface.
increasing ofinteractions
S1 concentration,
which
means
the
fluorescence
of
PFVCN
decreases
to
quenching
concentration was 0.5 U/mL, the ratio reached a plateau and did not increase further, which means
These results
areprobe
consistent
with the
resultsby
inS1.
Figure 1b. Figure 2b demonstrates
the relationship of
the ssDNA
was by
cleaved
by WS2 upon
ssDNA
analysis
S1.completely
The detection
limit was 5 ˆ 10´6 U/mL (the signal at the
the fluorescence intensity ratio (I0/I) of PFVCN to the concentration of S1, where I0 and I are the
detection limit
(Sdl ) is given
by:ofSsensor
where and
Sbl presence
is the signal
for a blank
without S1, σbl is
fluorescence
intensities
of S1 nuclease,
respectively.
dl = Ssolution
bl ´ 3 *inσthe
bl , absence
The ratio increases
with for
increasing
of S1 concentration,
which
the fluorescence
of PFVCN
the known standard
deviation
the blank’s
signal from
11 means
experiments.
In this
case, Sdl , Sbl , and
decreases due to quenching by WS2 upon ssDNA analysis by S1. The detection limit was
σbl are 275, 281,
and 1.5, respectively.), which is lower than that reported previously by Zhang et al.
5 × 10−6 U/mL (the signal at the detection limit (Sdl) is given by: Sdl = Sbl − 3 * σbl, where Sbl is the signal
´6 U/µL) [51], Zhou et al. (0.04 U/mL) [10] and Chu et al.
( 5 ˆ 10´5 U/mL)
[50],
Yuan
(1.4
ˆ 10
for a blank
without
S1,et
σblal.
is the
known
standard
deviation for the blank’s signal from 11 experiments.
´
7
In
this
case,
S
dl
,
S
bl
,
and
σ
bl
are
275,
281,
and
1.5,
respectively.),
which ssDNA
is lower than
reported oxide and
(5 ˆ 10 U/µL) [52]. He et al. studied the interaction
between
andthat
graphene
previously by Zhang et al. ( 5 × 10−5 U/mL) [50], Yuan et al. (1.4 × 10−6 U/μL) [51],
developed aZhou
GOetbased
biosensor for S1 nuclease with the detection limit of 5.8 ˆ 10´4 U/mL [53],
al. (0.04 U/mL) [10] and Chu et al. (5 × 10−7 U/μL) [52]. He et al. studied the interaction between
which is alsossDNA
higher
thatoxide
obtained
in thisa work.
When
theforS1S1concentration
0.5 U/mL, the
andthan
graphene
and developed
GO based
biosensor
nuclease with the was
detection
Figure −4
2. (a) Fluorescence emission spectra of PFVCN/ssDNA in Tris-HCl buffer solution (20 mM,
limit
of
5.8
×
10
U/mL
[53],
which
is
also
higher
than
that
obtained
in
this
work.
When
the
ratio reached a plateau
and did not increase further, which means the ssDNA probeS1was cleaved
pH 7.4) with the addition of S1; (b) Ratio of PFVCN fluorescence intensity with increasing amount of
concentration was 0.5 U/mL, the ratio reached a plateau and did not increase further, which means
completely by S1. S1 (Insert: plots of log[I /I] vs. log[S1]). [PFVCN] = 1.0 × 10 M, [ssDNA] = 10 nM, [WS ] = 1 μg/mL.
0
0
2
−6
dl
dl
bl
bl
bl
bl
dl
bl
bl
−5
−6
−7
−4
0
the ssDNAThe
probe
S1.
errorwas
barscleaved
representcompletely
the standard by
deviations
-6
2
of three parallel measurements. The excitation
wavelength is 470 nm.
Figure 2. (a) Fluorescence emission spectra of PFVCN/ssDNA in Tris-HCl buffer solution (20 mM,
Figure 2. (a)pH
Fluorescence
emission
spectra
PFVCN/ssDNA
in Tris-HCl
buffer
solution
(20 mM,
7.4) with the addition
of S1; (b)
Ratio ofof
PFVCN
fluorescence intensity
with increasing
amount
of
(Insert:
plots of
log[S1]).
1.0 × 10-6 M, [ssDNA]
= 10 nM,
[WS
2] = 1 μg/mL.
pH 7.4) withS1the
addition
oflog[I
S1;0/I]
(b)vs.Ratio
of [PFVCN]
PFVCN=fluorescence
intensity
with
increasing
amount of S1
The error bars represent the standard deviations of three parallel measurements. The excitation
(Insert: plots of log[I0 /I] vs. log[S1]). [PFVCN] = 1.0 ˆ 10-6 M, [ssDNA] = 10 nM, [WS2 ] = 1 µg/mL. The
wavelength is 470 nm.
error bars represent the standard deviations of three parallel measurements. The excitation wavelength
is 470 nm.
Sensors 2016, 16, 865
Sensors 2016, 16, 865
6 of 10
6 of 10
To study the selectivity of this method, we measured the fluorescence spectra of four enzymes,
study EcoRI,
the selectivity
of this
we measured
thesame
fluorescence
spectra of
four enzymes,
includingTo
ExoIII,
BSA and
KF method,
polymerase,
under the
experimental
conditions
as the S1
including
ExoIII,
EcoRI,
BSA
and
KF
polymerase,
under
the
same
experimental
conditions
as
the
measurement. As shown in Figure 3a, nearly negligible fluorescence change was observedS1in the
measurement. As shown in Figure 3a, nearly negligible fluorescence change was observed in the
presence
of ExoIII, EcoRI, BSA and KF polymerase relative to the control experiment (without any
presence of ExoIII, EcoRI, BSA and KF polymerase relative to the control experiment (without any
enzyme). The fluorescence intensity of PFVCN did not decrease dramatically after incubation with the
enzyme). The fluorescence intensity of PFVCN did not decrease dramatically after incubation with
otherthe
four
enzymes, which indicates that ssDNA cannot be cleaved by these enzymes. These results
other four enzymes, which indicates that ssDNA cannot be cleaved by these enzymes. These
indicate
that
the method
ismethod
specificisfor
S1. for S1.
results indicate
that the
specific
Figure 3. (a) Specificity of the assay toward other enzymes. [PFVCN] = 1.0 × 10−6 M, [WS2] = 1μg/mL,
Figure
3. (a) Specificity of the assay toward other enzymes. [PFVCN] = 1.0 ˆ 10´6 M, [WS ] = 1µg/mL,
[ssDNA] = 10 nM, [S1] = [EcoRI] = 0.5 U/mL, [KF Polymerase] = [ExoIII] = 5 U/mL, [BSA] = 0.012mg/mL;
[ssDNA]
= 10 nM, efficiency
[S1] = [EcoRI]
0.5 U/mL,
[KFin
Polymerase]
= [ExoIII]
= 5(20
U/mL,
[BSA]
0.01 mg/mL;
(b) Inhibition
of S1 =
nuclease
by ATP
Tris-HCl buffer
solution
mM, pH
7.4).=[PFVCN]
(b) Inhibition
S1 nuclease
byU/mL,
ATP [ssDNA]
in Tris-HCl
buffer
mM, pH
= 1.0 × 10−6efficiency
M, [WS2] =of
1 μg/mL,
[S1] = 0.5
= 10 nM.
Thesolution
error bars(20
represent
the 7.4).
[PFVCN]
= 1.0deviations
ˆ 10´6 M,
[WS2parallel
] = 1 µg/mL,
[S1] =The
0.5excitation
U/mL, [ssDNA]
nM.
standard
of three
measurements.
wavelength= is10
470
nm.The error bars
represent the standard deviations of three parallel measurements. The excitation wavelength is 470 nm.
Furthermore, S1 reaction time was optimized. A series of reaction mixtures containing a fixed
concentration of 10 nM ssDNA probe and 0.5 U/mL S1 nuclease was incubated at 37 °C for 0, 5, 10,
Furthermore, S1 reaction time was optimized. A series of reaction mixtures containing a fixed
15, 20, 25, 30, or 35 min. As shown in Figure S4, the fluorescence intensity of PFVCN gradually
concentration of 10 nM ssDNA probe and 0.5 U/mL S1 nuclease was incubated at 37 ˝ C for 0, 5,
decreased as a function of time. The fluorescence intensity rapidly decreased during the first 5 min.
10, 15,
20,fluorescence
25, 30, or 35
min. As
shown almost
in Figure
S4, the
fluorescence
intensity
of PFVCN
gradually
The
intensity
remained
constant
after
25 min, which
demonstrates
that the
S1
decreased
as
a
function
of
time.
The
fluorescence
intensity
rapidly
decreased
during
the
first
5 min.
nuclease detection can be completed in a short time.
The fluorescence
intensity
remained
almost
constant
25 min,
which
demonstrates
that the S1
In addition,
we further
studied the
inhibition
of theafter
enzymatic
activity
of S1.
ATP, a well-known
nuclease
detection
can bewas
completed
in a short
time.
S1 nuclease
inhibitor,
used to inhibit
the enzymatic
activity of S1. The concentration of S1 was
fixed
at 0.5 U/mL.
Figure 3b
showsthe
the inhibition
inhibition efficiency
of ATP varied
in the
range
In
addition,
we further
studied
of the enzymatic
activity
ofconcentration
S1. ATP, a well-known
from
0
to
70
μM
(I
0
and
I
are
the
fluorescence
intensities
of
the
sensor
solution
in
the
absence
S1 nuclease inhibitor, was used to inhibit the enzymatic activity of S1. The concentration ofand
S1 was
of ATP).
The activity
of S1
was
inhibited efficiency
by the addition
of varied
30 μM ATP.
results
fixedpresence
at 0.5 U/mL.
Figure
3b shows
the
inhibition
of ATP
in theThe
concentration
demonstrate that the developed method demonstrates strong performance not only in the assay of
range from 0 to 70 µM (I0 and I are the fluorescence intensities of the sensor solution in the absence
endonucleases activity but also in the screening of endonucleases inhibitors, which is of great
and presence of ATP). The activity of S1 was inhibited by the addition of 30 µM ATP. The results
importance in modern drug discovery.
demonstrate that the developed method demonstrates strong performance not only in the assay
of endonucleases
activity Radicals
but also in the screening of endonucleases inhibitors, which is of great
3.4. Sensing of Hydroxyl
importance in modern drug discovery.
Hydroxyl radicals can damage virtually all types of macromolecules, such as carbohydrates,
nucleic acids and lipids; this damage is highly dangerous and toxic to organisms. Assays for DNA
3.4. Sensing of Hydroxyl Radicals
cleavage by hydroxyl radicals can be performed by using a similar approach with the addition of
Fenton’s reagent
(Fe2+can
+ H2damage
O2). Fenton’s
reagentall
generates
radicals thatsuch
randomly
cut DNA
Hydroxyl
radicals
virtually
types ofhydroxyl
macromolecules,
as carbohydrates,
2+]:[H2O2]:[DTT] =
intoacids
different
fragments
andiseven
single
bases. Fenton’s
reagent
nucleic
andsequence
lipids; this
damage
highly
dangerous
and toxic
to ([Fe
organisms.
Assays for
1:10:10)
was
chosen
here
to
generate
hydroxyl
radicals.
To
study
the
activity
of
hydroxyl
radicals,
we the
DNA cleavage by hydroxyl radicals can be performed by using a similar approach with
measured the fluorescence spectra
in 20 mM Tris-HCl buffer at pH 7.0 ([PFVCN] =1.0 × 10−6 mol/L in
2+
addition of Fenton’s reagent
(Fe + H2 O2 ). Fenton’s reagent generates hydroxyl radicals that
RUs, [ssDNA] = 1.0 × 10−8 mol/L, [WS2] = 1 μg/mL) at various concentrations of Fe2+ (from 0.01 μM to
randomly cut DNA into different sequence fragments and even single bases. Fenton’s reagent
5 μM). Figure 4a shows that the fluorescence of PFVCN gradually decreases with the increasing of
([Fe2+Fe
]:[H
1:10:10)
wasfrom
chosen
to generate
hydroxyl
radicals.
To study
theand
activity
2 O2 ]:[DTT] =which
2+ concentration,
results
the here
nucleotide
acids being
cleaved
by hydroxyl
radicals
of hydroxyl radicals, we measured the fluorescence spectra in 20 mM Tris-HCl buffer at pH 7.0
([PFVCN] =1.0 ˆ 10´6 mol/L in RUs, [ssDNA] = 1.0 ˆ 10´8 mol/L, [WS2 ] = 1 µg/mL) at various
concentrations of Fe2+ (from 0.01 µM to 5 µM). Figure 4a shows that the fluorescence of PFVCN
Sensors 2016, 16, 865
7 of 10
gradually decreases with the increasing of Fe2+ concentration, which results from the nucleotide acids
beingSensors
cleaved
radicals and gradually being reduced by Fenton’s reagent. Figure 74b
shows
2016,by
16, hydroxyl
865
of 10
2+
that the fluorescence intensity ratio (I0 /I) of PFVCN increases with the increasing of Fe concentration,
gradually
reduced
by Fenton’s
reagent.of
Figure
4b shows
that theinfluorescence
ratio of
where
I0 and Ibeing
are the
fluorescence
intensities
the sensor
solution
the absenceintensity
and presence
(I0/I) reagent,
of PFVCNrespectively.
increases withThe
the increasing
of Fe2+of
concentration,
where I0was
and Irelated
are the fluorescence
Fenton’s
concentration
hydroxyl radicals
to Fe2+ , and the
intensities of the sensor solution in the absence and presence of Fenton’s reagent, respectively. The
detection
limit was 0.01 µM. The result indicates that our new platform also can be used to detect
concentration of hydroxyl radicals was related to Fe2+, and the detection limit was 0.01 μM. The result
hydroxyl radicals sensitively and simply.
indicates that our new platform also can be used to detect hydroxyl radicals sensitively and simply.
Figure 4. (a) Fluorescence emission spectra of PFVCN/ssDNA in Tris-HCl buffer solution (20 mM,
Figure
4. (a) Fluorescence emission spectra of PFVCN/ssDNA in Tris-HCl buffer solution (20 mM,
pH 7.0) with the addition of Fenton reagent ([Fe2+]:[H2O2]:[DTT] = 1:10:10); (b) Ratio of PFVCN
2+
pH 7.0)
with theintensity
additionwith
of Fenton
reagent
([Fe
(b) Ratio
of nM,
PFVCN
2+. [PFVCN]
2 O2 ]:[DTT]
fluorescence
increasing
amount
of Fe]:[H
= 1.0=×1:10:10);
10−6 M, [ssDNA]
= 10
2+ . [PFVCN] = 1.0 ˆ 10´6 M, [ssDNA] = 10 nM,
fluorescence
intensity
with
increasing
amount
of
Fe
[WS2] = 1 μg/mL. The error bars represent the standard deviations of three parallel measurements.
[WS2The
] = 1excitation
µg/mL.wavelength
The error bars
the standard deviations of three parallel measurements.
is 470represent
nm.
The excitation wavelength is 470 nm.
Some antioxidants display effective capabilities to scavenge ·OH; therefore, they are able to
inhibit the cleavage of DNA by OH. Thiourea was selected to clean up hydroxyl radicals in this case.
Some antioxidants display effective capabilities to scavenge ¨ OH; therefore, they are able to inhibit
As shown in Figure S5, the inhibition efficiency of thiourea varied in the concentration range from 0
the cleavage
of DNA by OH. Thiourea was selected to clean up hydroxyl radicals in this case. As shown
to 0.7 mM, whereas the concentration of Fe2+ was fixed at 5 μM (I0 and I are the fluorescence intensities
in Figure
S5,
the inhibition
thiourea
varied
in the concentration
from
0 to 0.7 mM,
of the sensor
solution inefficiency
the absenceofand
presence
of thiourea).
The activity ofrange
·OH was
effectively
2+ was fixed at 5 µM (I and I are the fluorescence intensities of the
whereas
the
concentration
of
Fe
inhibited at 0.5 mM. Therefore, the developed platform0provides a rapid and convenient method to
sensor
solution
in the absence
and presence
of thiourea). The activity of ¨ OH was effectively inhibited
screen
anti-oxidant
natural products
or drugs.
at 0.5 mM. Therefore, the developed platform provides a rapid and convenient method to screen
4. Conclusions
anti-oxidant
natural products or drugs.
In summary, we have designed a new method to detect S1 nuclease and hydroxyl radicals by
4. Conclusions
taking advantage of the new platform based on PFVCN and WS2 nanosheets. This strategy has three
significant characteristics. First, it is sensitive with a detection limit of S1 5 × 10 U/mL, which is far
In
summary, we have designed a new method to detect S1 nuclease and hydroxyl radicals by
lower than reported in the literature. Second, the assay is cost-efficient without the need for
taking advantage of the new platform based on PFVCN and WS2 nanosheets. This strategy has three
dye-labelling of ssDNA due to the use of economic WS2 as the acceptor. Third, this method
is simple
significant
characteristics. First, it is sensitive with a detection limit of S1 5 ˆ 10´6 U/mL, which
and rapid, avoiding complicated washing and separation procedures. In virtue of these advantages,
is farthe
lower
than reported
in the literature.
Second,
the assay
is cost-efficient
without provides
the need for
proposed
strategy combining
water-soluble
conjugated
polymer
and WS2 nanosheets
dye-labelling
of ssDNA
dueoftobiosensors
the use offor
economic
WS2 detection
as the acceptor.
method
is simple
new insight
in the area
endonuclease
and the Third,
sensingthis
of other
reactive
and rapid,
oxygenavoiding
species. complicated washing and separation procedures. In virtue of these advantages,
the proposed
strategy
combining
water-soluble
polymer and WS
provides
2 nanosheets
Supplementary
Materials:
The following
are availableconjugated
online at www.mdpi.com/link,
Figure
S1: TEM image
of
new insight
in the
areafluorescence
of biosensors
detection
and theS1/WS
sensing
of other reactive
WS2, Figure
S2: The
spectrafor
of endonuclease
PFVCN/ssDNA/WS
2, PFVCN/ssDNA/
2, PFVCN/ssDNA
,
PFVCN/ssDNA/OH/WS
2, Figure S3: Absorption and emission spectra of WS2, and PFVCN/WS2 in Tris-HCl
oxygen
species.
−6
buffer solution, Figure S4: Fluorescence intensity of PFVCN/ssDNA in the presence of S1 nuclease incubated for
different periods
in Tris-HCl
buffer solution,
Figure S5:
Inhibition
efficiency of hydroxyl radical by thiourea in
Supplementary
Materials:
The following
are available
online
at http://www.mdpi.com/1424-8220/16/6/865/s1,
buffer
solution.
FigureTris-HCl
S1: TEM
image
of WS2 , Figure S2: The fluorescence spectra of PFVCN/ssDNA/WS2 , PFVCN/ssDNA/
S1/WS2 , PFVCN/ssDNA , PFVCN/ssDNA/OH/WS2 , Figure S3: Absorption and emission spectra of WS2 , and
Acknowledgments: We are grateful for the financial support from the National Natural Science Foundation of
PFVCN/WS2 in Tris-HCl buffer solution, Figure S4: Fluorescence intensity of PFVCN/ssDNA in the presence of
China (Grants
21222509),
the Program
forin
Changjiang
and Innovative
Team in
University
S1 nuclease
incubated
for different
periods
Tris-HCl Scholars
buffer solution,
FigureResearch
S5: Inhibition
efficiency
of(IRT
hydroxyl
14R33),
and thein
Program
forbuffer
Innovative
Research Team in Shaanxi Province (No. 2014KCT-28).
radical
by thiourea
Tris-HCl
solution.
Sensors 2016, 16, 865
8 of 10
Acknowledgments: We are grateful for the financial support from the National Natural Science Foundation of
China (Grants 21222509), the Program for Changjiang Scholars and Innovative Research Team in University
(IRT 14R33), and the Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28).
Author Contributions: All authors contributed extensively to the work in this paper. Yanli Tang and Junting Li
conceived and designed the experiments; Junting Li performed the experiments; Junting Li, Qi Zhao and Yanli
Tang analyzed the data; Junting Li and Yanli Tang wrote the paper. All authors read and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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