sensors
Article
Potentiometric Aptasensing of Vibrio alginolyticus
Based on DNA Nanostructure-Modified
Magnetic Beads
Guangtao Zhao 1,2,† , Jiawang Ding 1,† , Han Yu 1,2 , Tanji Yin 1, * and Wei Qin 1, *
1
2
*
†
Key Laboratory of Coastal Environmental Processes and Ecological Remediation,
Yantai Institute of Coastal Zone Research (YIC), Chinese Academy of Sciences (CAS);
Shandong Provincial Key Laboratory of Coastal Environmental Processes, YICCAS, Yantai 264003,
Shandong, China; [email protected] (G.Z.); [email protected] (J.D.); [email protected] (H.Y.)
University of Chinese Academy of Sciences, Beijing 100049, China
Correspondence: [email protected] (T.Y.); [email protected] (W.Q.); Tel.: +86-535-210-9156 (W.Q.)
These authors contributed equally to this work.
Academic Editor: Huangxian Ju
Received: 23 September 2016; Accepted: 28 November 2016; Published: 2 December 2016
Abstract: A potentiometric aptasensing assay that couples the DNA nanostructure-modified
magnetic beads with a solid-contact polycation-sensitive membrane electrode for the detection
of Vibrio alginolyticus is herein described. The DNA nanostructure-modified magnetic beads are
used for amplification of the potential response and elimination of the interfering effect from a
complex sample matrix. The solid-contact polycation-sensitive membrane electrode using protamine
as an indicator is employed to chronopotentiometrically detect the change in the charge or DNA
concentration on the magnetic beads, which is induced by the interaction between Vibrio alginolyticus
and the aptamer on the DNA nanostructures. The present potentiometric aptasensing method shows
a linear range of 10–100 CFU mL−1 with a detection limit of 10 CFU mL−1 , and a good specificity
for the detection of Vibrio alginolyticus. This proposed strategy can be used for the detection of other
microorganisms by changing the aptamers in the DNA nanostructures.
Keywords: potentiometric aptasensing; Vibrio alginolyticus; DNA nanostructures; magnetic
beads; protamine
1. Introduction
As an opportunistic marine pathogen, Vibrio alginolyticus (V. alginolyticus) can not only lead to
septicemias and corneal opaqueness in fish and shell diseases and white spots in shrimps [1–3], but
also infects mammalian cells causing otitis, wound infection, and chronic diarrhea [4,5]. The rapid
and sensitive detection of V. alginolyticus is of great importance for aquaculture, food industry, and
clinical diagnosis.
The agar plate culture method and enzyme-linked immunosorbent assays are commonly used for
the detection of bacteria. However, they suffer from such problems as time-consuming procedures,
false-positive signals, low sensitivity, and poor specificity [6]. The polymerase chain reaction (PCR)
technique, especially for the multiplex PCR, has been developed for the detection of bacteria with
good specificity and high sensitivity [7–10]. However, this technique needs complex pretreatment
procedures and expensive instruments.
Aptamers are single-stranded DNA or RNA oligonucleotides designed through the systematic
evolution of ligands by exponential enrichment. Since aptamers have high specificities and binding
affinities to their targets, they have been widely used to develop a variety of electrochemical biosensors
for the detection of small molecules, metal ions, and proteins [11–15]. Nowadays, aptamers have
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also been used for the detection of bacteria based on their high specificity bindings to proteins
either on the cell surfaces or inside the cells [16–19]. Various electrochemical aptasensors using
different transduction modes such as amperometry, impedimetry, and potentiometry have been
developed [20–22]. Recently, our group developed a label-free potentiometric aptasensing method for
the detection of Listeria monocytogenes in a homogeneous solution [23], based on the polycation-sensitive
membrane electrode using protamine as an indicator. However, for that method, other bacteria
with negative charges may interfere with the potential response of protamine. Additionally, the
polycation-sensitive membrane electrode may suffer from problems of strong potential drifts, since the
extraction of polycation into the membrane is an irreversible process. The purpose of this work was to
develop a new method for the potentiometric detection of bacteria, which could not only eliminate
sample interferences but also show stable and reversible potential responses.
Herein, we present a novel potentiometric aptasensing strategy for the detection of V. alginolyticus
based on DNA nanostructure-modified magnetic beads for signal amplification and magnetic
separation, and on a solid-contact polycation-sensitive membrane electrode for reversible potential
detection via chronopotentiometry. In the presence of V. alginolyticus, the DNA nanostructure-modified
magnetic beads can be disassembled, which is induced by the specific binding interactions between the
target bacterial cells and the aptamer molecules in the DNA nanostrutures. After magnetic separation,
the change in the charge or DNA concentration on the magnetic beads can be chronopotentiometrially
detected by the solid-contact polycation-sensitive electrode using protamine as an indicator based on
the electrostatic interaction between DNA and protamine.
2. Materials and Methods
2.1. Materials
High-molecular-weight poly(vinyl chloride) (PVC), 2-nitrophenyl octyl ether (o-NPOE),
tetradodecylammonium chloride (TDDACl), dinonylnaphthalene sulfonic acid (DNNS, 50 wt %
solution in heptanes), tris (hydroxymethyl)-aminomethane (Tris), protamine sulfate salt, and ionic
liquid (IL, 1-butyl-3-methyl-imidazolium tetrafluoroborate) were purchased from Sigma-Aldrich.
Carboxylic-multiwall carbon nanotubes (MWCNTs) were obtained from Nanjing XFNANO Materials
Technology Co. Ltd. (Nanjing, China). The lipophilic salt DNNS-TDDA was synthesized as described
before [24]. The magnetic beads (1 µm in diameter) were purchased from Bio Canal Co. Ltd.
(Wuxi, China). Bacterial strains for V. alginolyticus 1.1587 and Aeromonas hydrophila 1.172 were purchased
from Yudingxinjie Technology Co. Ltd. (Beijing, China). Bacterial strains for Escherichia coli (E. coli)
ATCC 27853 and Staphylococcus aureus 08032813 were kindly provided from the Biotechnology Lab
of Binzhou Medical University. The number of colony-forming units per mL (CFU mL−1 ) for each
bacteria culture was determined by the surface plate counting method. The aptamer used in this work
for the detection of V. alginolyticus was selected by Zheng et al. [3]. The sequences of the aptamer
DNA and other DNA are shown in Table 1. All the sequences were synthesized by Shanghai Sangon
Biotechnology Co. Ltd. (Shanghai, China). The buffer for DNA hybridization contained 100 mM NaCl,
5 mM KCl, 50 mM Tris-HCl, and 1 mM MgCl2 (pH = 7.4).
Table 1. Sequences of the oligonucleotides used in this work.
Oligonucleotide
Sequence
Aptamer DNA
50 -TCAGTCGCTTCGCCGTCTCCTTCAGCCGGGGTGGTCAGTAGGA
GCAGCACAAGAGGGAGACCCCAGAGGG-30 [3]
Capture DNA
50 -TTTTTCCCTCTGGGGTCTCCC-30 (modified with biotin at 50 terminal end)
H 1 DNA
50 -CGGCGAAGCGACTGACAAAGTCTAGTCGCT-30
H 2 DNA
50 -TCAGTCGCTTCGCCGAGCGACTAGACTTTG-30
Random DNA
50 -GAGTAGTTCGTG GCCTAG-30
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2.2. Synthesis of the DNA Nanostructure-Modified Magnetic Beads
All the oligonucleotides were kept at 95 ◦ C for 2 min and then cooled at room temperature for
1 h before use [25–27]. The DNA nanostructure-modified magnetic beads were prepared as described
before [28]. The capture DNA modified magnetic beads were obtained by incubating 150 µL of
50 -biotin-modified capture DNA fragments (3 µM) with 150 µL of streptavidin modified magnetic
beads (10 mg·mL−1 ) via shaking for 0.5 h. After magnetic separation and washing with buffer, the
capture DNA modified magnetic beads were incubated with 150 µL of aptamer DNA fragments (3 µM)
via shaking for 1 h, and the capture/aptamer DNA modified magnetic beads were thus obtained.
The H1/H2 DNA complexes were prepared by incubating 150 µL of H1 DNA fragments (3 µM) with
150 µL of H2 DNA fragments (3 µM) via shaking for 1 h. The H1/H2 DNA complexes (150 µL) were
then incubated with 150 µL of capture/aptamer DNA modified magnetic beads with 1 h shaking.
After magnetic separation and washing with buffer, the DNA nanostructure-modified magnetic beads
were obtained and re-dispersed in 150 µL of buffer solution. For the detection of bacteria, 40 µL of the
prepared DNA nanostructure-modified magnetic bead solution (10 mg·mL− 1 ) were used.
2.3. Preparation of the Solid-Contact Polycation-Sensitive Electrode
Prior to modification, the glassy carbon electrode (GCE, 3 mm in diameter) was polished with
emery paper and alumina slurries followed by rinsing thoroughly with ultrapure water. The electrode
was successively ultrasonicated in water and ethanol, and then allowed to dry at room temperature.
A mixture of 5 mg of MWCNTs and 1 µL of IL was ground in an agate mortar for 10 min [29], and
then dispersed in 1 mL of ultrapure water with ultra-sonication to obtain a 5 mg·mL−1 suspension
solution. Twenty microliters of the MWCNT-IL suspension solution was coated onto the surface of the
GCE and allowed to dry under an infrared lamp.
The polycation-sensitive membrane contained 10.0 wt % DNNS-TDDA, 60.0 wt % o-NPOE, and
30.0 wt % PVC. One hundred milligrams of the membrane components were dissolved in 800 µL of
tetrahydrofuran (THF). Eighty microliters of the membrane solution were coated onto the MWCNT-IL
modified GCE. After the evaporation of THF, the prepared solid-contact polycation-sensitive electrodes
were conditioned in 10 mM NaCl for at least 12 h before use, and kept in the conditioning solution
when not in use.
2.4. Determination of V. alginolyticus via Chronopotentiometry
Forty microliters of DNA nanostructure-modified magnetic beads were mixed with 50 µL of
V. alginolyticus at different concentrations at room temperature for 30 min. After magnetic separation,
the resulting magnetic beads were then incubated with 7.5 µL of 1 mg·mL−1 protamine. The unreacted
protamine after magnetic separation was added into 1.5 mL of 0.01 M NaCl and detected by the
solid-contact polycation-sensitive electrode via chronopotentiometry.
Chronopotentiometry was performed by using a CHI-760C electrochemical workstation
(Chenhua Corp., Shanghai, China) with a conventional three-electrode system comprising the proposed
solid-contact polycation-sensitive membrane electrode as a working electrode, a platinum wire as
counter electrode, and an Ag/AgCl electrode (saturated KCl) as a reference electrode. During the
chronopotentiometric experiments, an external cathodic current of 5 µA with a duration of 1 s was used
to extract protamine into the polycation-sensitive membrane. The solid-contact polycation-sensitive
membrane electrode was refreshed under a controlled voltage at the open-circuit potential in
the absence of protamine with a recovery time of 180 s for multiple consecutive measurements.
The difference between the potentials measured at 0.5 s after applying the current in the absence and
presence of V. alginolyticus was used for quantification.
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3. Results
Results and
and Discussion
Discussion
3.
3.1.
SensingPrinciple
Principle
3.1. Sensing
Figure
principle
forfor
thethe
potentiometric
aptasensing
of V.of
alginolyticus
basedbased
on DNA
Figure 11shows
showsthe
the
principle
potentiometric
aptasensing
V. alginolyticus
on
nanostructure-modified
magnetic
beads.beads.
The capture
DNADNA
was immobilized
on the
of the
DNA nanostructure-modified
magnetic
The capture
was immobilized
onsurface
the surface
of
magnetic
beadsbeads
via thevia
strong
streptavidin-biotin
interaction.
The aptamer
H1/H2
DNA
hybridize
the magnetic
the strong
streptavidin-biotin
interaction.
The and
aptamer
and
H1/H2
DNA
successively
with the capture
DNA
through
complementary
base-pairing
reactions reactions
to form the
hybridize successively
with the
capture
DNAthe
through
the complementary
base-pairing
to
DNA
nanostructure-modified
magneticmagnetic
beads. In
the presence
of theof
target,
the aptamer
on the
form the
DNA nanostructure-modified
beads.
In the presence
the target,
the aptamer
on
DNA
nanostructure-modified
magnetic
beads
specifically
binds
totothe
the DNA
nanostructure-modified
magnetic
beads
specifically
binds
thetarget,
target,which
whichleads
leads to
to the
the
disassembly
DNA
nanostructures
and subsequently
influencesinfluences
the charge the
or DNA
concentration
disassemblyofofthethe
DNA
nanostructures
and subsequently
charge
or DNA
on
the magnetic
[28]. This
aptamer/target
binding eventbinding
can be detected
solid-contact
concentration
onbeads
the magnetic
beads
[28]. This aptamer/target
event canbybethe
detected
by the
polycation-sensitive
electrode using electrode
protamine using
as an indicator,
based
electrostatic
interaction
solid-contact polycation-sensitive
protamine
as on
an the
indicator,
based
on the
between
the DNA
nanostructures
andDNA
protamine.
electrostatic
interaction
between the
nanostructures and protamine.
Figure 1.
1. Schematic
alginolyticus based
based on
on DNA
DNA
Figure
Schematic illustration
illustration of
of potentiometric
potentiometric aptasensing
aptasensing of
of V.
V. alginolyticus
nanostructure-modified
magnetic
beads
for
(A)
a
control
and
(B)
a
given
target
assay.
nanostructure-modified magnetic beads for (A) a control and (B) a given target assay.
Figure 2 shows the principle for the detection of protamine with the polycation-sensitive
Figure 2 shows the principle for the detection of protamine with the polycation-sensitive electrode
electrode based on the MWCNT-IL composite as solid contact via chronopotentiometry. The
based on the MWCNT-IL composite as solid contact via chronopotentiometry. The application of
application of the imidazolium-based IL is to prevent the formation of agglomerates of MWCNTs
the imidazolium-based IL is to prevent the formation of agglomerates of MWCNTs through cation-π
through cation-π interactions between the imidazolium cation and the π-electrons of nanotubes [30],
interactions between the imidazolium cation and the π-electrons of nanotubes [30], while MWCNTs are
while MWCNTs are used to decrease the charge transfer resistance and improve the electrical
used to decrease the charge transfer resistance and improve the electrical conductivity at the interface
conductivity at the interface between the GCE and the polycation-sensitive membrane [31]. Under
between the GCE and the polycation-sensitive membrane [31]. Under zero current conditions, there is
zero current conditions, there is no potential response to protamine, due to the presence of the
no potential response to protamine, due to the presence of the neutral lipophilic salt DNNS-TDDA in
neutral lipophilic salt DNNS-TDDA in the polycation-sensitive membrane. When a constant
the polycation-sensitive membrane. When a constant cathodic current is applied, there is a net flux of
cathodic current is applied, there is a net flux of polycation in the direction of the membrane phase,
polycation in the direction of the membrane phase, since protamine can electrostatically interact with
since protamine can electrostatically interact with DNNS in the membrane phase to form the
DNNS in the membrane phase to form the cooperative ion pairs [32]. Additionally, in order to obtain
cooperative ion pairs [32]. Additionally, in order to obtain the reversible potential response,
the reversible potential response, protamine can be removed from the polycation-sensitive membrane
protamine can be removed from the polycation-sensitive membrane by applying a controlled
by applying a controlled voltage at the open-circuit potential.
voltage at the open-circuit potential.
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Figure 2. Schematic diagram of the polycation-sensitive electrode based on a MWCNT-IL composite
Figure
Schematic
diagram
thepolycation-sensitive
polycation-sensitive
electrode
based
a MWCNT-IL
composite
Figure
2.2. Schematic
ofofthe
electrode
based
onon
a MWCNT-IL
composite
as
as a solid
contact fordiagram
the chronopotentiometric
detection
of protamine.
a solid
contact
chronopotentiometric
detection
of protamine.
aassolid
contact
for for
the the
chronopotentiometric
detection
of protamine.
3.2. Optimization of Protamine Concentration
3.2. Optimization
Optimization of
of Protamine
Protamine Concentration
Concentration
3.2.
Since protamine was used as an indicator to detect the change in the DNA concentration on the
Since
protamine was
as
change
in in
thethe
DNA
on the
protamine
was used
usedmagnetic
asan
anindicator
indicator
todetect
detectthe
the
DNA
concentration
on
DNASince
nanostructure-modified
beadstoinduced
by
V.change
alginolyticus,
the concentration
effect
of protamine
DNA
nanostructure-modified
beads
induced
bybyV.V.alginolyticus,
the
protamine
the
DNA
nanostructure-modified
magnetic
beads
alginolyticus,
the effect
effect
of
protamine
concentration
was investigated.magnetic
Figure
3A
show
sinduced
the potentiometric
responses
of theof
solid-contact
concentration
was investigated.
investigated.
Figure 3A
3Aunder
showan
the
potentiometric
responses
of
the
solid-contact
concentration
was
Figure
show
ss the
potentiometric
responses
the
solid-contact
polycation-sensitive
membrane electrode
external
cathodic current
of 5of
μA
with
a duration
polycation-sensitive
membrane
electrode
under
an
external
cathodic
current
of
5
μA
with
duration
polycation-sensitive
electroderesponse
under anofexternal
cathodic current of
5 µA withelectrode
aa duration
of 1 s. It can be seenmembrane
that the potential
the polycation-sensitive
membrane
in
of
1
s.
It
can
be
seen
that
the
potential
response
of
the
polycation-sensitive
membrane
electrode
in
of
1
s.
It
can
be
seen
that
the
potential
response
of
the
polycation-sensitive
membrane
electrode
in
the
the presence of protamine is larger than that in the absence of protamine, which indicates that
the
presence
of protamine
is
larger
than
thatabsence
in themembrane.
absence
ofFigure
protamine,
which
indicates
presence
of protamine
isinto
larger
that
in the
of
protamine,
which
that
protamine
protamine
is extracted
thethan
polycation-sensitive
3Bindicates
shows
the
curve
ofthat
the
protamine
is extracted
intomembrane
the polycation-sensitive
membrane.
Figure
3B
curve
of the
is
extractedchange
into
the of
polycation-sensitive
membrane.vs.
Figure
shows
the curve
of the the
potential
change
potential
the
electrode
the 3B
concentration
of shows
protamine.
When
the
potential
change
of the vs.
membrane
electrode
vs. the change
concentration
When the
−1, the of
of
the membrane
the5 concentration
protamine.
Whenisthe
of protamine
is
concentration
ofelectrode
protamine
is
μg·mL
potential
upconcentration
toof30protamine.
mV. Considering
the
−1, the potential change is up to 30 mV. Considering the
−
1
concentration
of
protamine
is
5
μg·mL
5subsequent
µg·mL , the
potential change
is up to
30 mV. the
Considering
the subsequentand
electrostatic
interaction
electrostatic
interaction
between
DNA nanostructures
protamine
and the
subsequent
interactionand
between
theelectrode,
DNAthe
nanostructures
and
protamine
and the
between
theofelectrostatic
DNA
nanostructures
protamine
and
sensitivity
the polycation-sensitive
sensitivity
the polycation-sensitive
membrane
5 μg·mL−1 of
protamine
was selected
for
−1 protamine was selected for
−
1
sensitivity
of
the
polycation-sensitive
membrane
electrode,
5
μg·mL
membrane
electrode, 5 µg·mL protamine was selected for further experiments.
further experiments.
further experiments.
Figure 3.
Potentiometric
responses
of theofsolid-contact
polycation-sensitive
membrane
electrode
Figure
3. (A)(A)
Potentiometric
responses
the solid-contact
polycation-sensitive
membrane
Figure
3.
(A)
Potentiometric
responses
of
the
solid-contact
polycation-sensitive
membrane
electrode
in 1.5 mLin
of1.5
0.01mL
M of
sodium
different
concentrations
of (a) 5; (b)of3;
electrode
0.01 Mchloride
sodiumsolution
chloridewith
solution
withprotamine
different protamine
concentrations
−1. (B) Plot
in
1.5
mL0.5;
of 0.01
M
sodium
chloride
solution
with
different
protamine
concentrations
of (a) 5; (b) 3;
−
1
shows
potential
changes
over
the
protamine
concentration
(c)
2;
(d)
and
(e)
0
μg·mL
(a–e) 5; 3; 2; 0.5; 0 µg·mL . (B) Plot shows potential changes over the protamine concentration range
−1
Plot shows
potentialdeviation
changes for
over
the measurements.
protamine concentration
(c)
2; (d)
0.5;−
and
(e)−1.0Error
μg·mL
1 . Error
range
of ·0–5
bars. (B)
represent
one standard
three
of
0–5 µg
mL μg·mL
bars represent
one standard
deviation for three
measurements.
−1
range of 0–5 μg·mL . Error bars represent one standard deviation for three measurements.
3.3. Optimization
Optimization of
of the
the Amount
Amountofofthe
theDNA
DNANanostructure-Modified
Nanostructure-ModifiedMagnetic
MagneticBeads
Beads
3.3.
3.3. Optimization of the Amount of the DNA Nanostructure-Modified Magnetic Beads
The influence
influence of
of the
the amount
amount of
of the
the DNA
DNA nanostructure-modified
nanostructure-modified magnetic
magnetic beads
beads on
on the
the
The
The
influence
of
the
amount
of
the
DNA
nanostructure-modified
magnetic
beads
on
the
potential response
response to
to protamine
protamine of
ofthe
thesolid-contact
solid-contactpolycation-sensitive
polycation-sensitivemembrane
membrane electrode
electrode was
was
potential
potential
response
to protamine
of4A,
thethere
solid-contact
polycation-sensitive
membrane
electrode
was
investigated.
As
shown
in
Figure
is
an
obvious
potential
change
in
the
presence
of
the
investigated. As shown in Figure 4A, there is an obvious potential change in the presence of the
investigated.
As
shown
in
Figure
4A,
there
is
an
obvious
potential
change
in
the
presence
of
the
DNA nanostructure-modified magnetic beads, which is due to the electrostatic interaction between
DNA
nanostructure-modified
magnetic beads,
which isasdue
to theinelectrostatic
interaction
the DNA
nanostructures and protamine.
Additionally,
shown
Figure 4B, the
potentialbetween
change
the DNA nanostructures and protamine. Additionally, as shown in Figure 4B, the potential change
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DNA
magnetic beads, which is due to the electrostatic interaction between
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2016, 16, 2052
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the
DNA
nanostructures
and
protamine.
Additionally, asmagnetic
shown inbeads
Figure
4B, the potential
change
increases as the amount of DNA nanostructure-modified
increases
up to 40 μL,
and
increases
as
the
amount
of
DNA
nanostructure-modified
magnetic
µL,
and
beads
increases
up
to
40
μL,
thereafter remains almost constant. Therefore, a volume of 40 μL was used as the amount of the
thereafter
remains
Therefore,
volume
ofstudy.
40
usedused
as the
of theof
DNA
thereafter
remainsalmost
almostconstant.
constant.
Therefore,
a further
volume
of µL
40 was
μL was
asamount
the amount
the
DNA
nanostructure-modified
magnetic
beadsafor
nanostructure-modified
magnetic
beads
for
further
study.
DNA nanostructure-modified magnetic beads for further study.
Figure 4. (A) Potentiometric responses of the solid-contact polycation-sensitive membrane electrode
Figure
4. (A)
(A)
Potentiometric
responses
of the
the solid-contact
solid-contact
polycation-sensitive
membrane
electrode
−1 protamine in the
in
1.5 mL
of Potentiometric
0.01
M sodiumresponses
chloride solution
with 5 μg·mL
presenceelectrode
of DNA
Figure
4.
of
polycation-sensitive
membrane
−1
−
1
in 1.5
1.5 mL
mL of
of 0.01
0.01 M
M sodium
sodium
chloride
solution
with 55µg
μg·mL
protamine
in
the
presence
of DNA
DNA
nanostructure-modified
magnetic
beads
with different
volumes
(a) 0; (b) 5;
10;presence
(d) 30; and
(e)
40
in
chloride
solution
with
·mL of
protamine
in(c)
the
of
nanostructure-modified
beads
with
(a)(a–e)
0; (b)
30;
and
(e)
40
nanostructure-modified
magnetic
beads
withdifferent
different
volumes
5;(c)
10;10;
30;(d)
40
µL.
(B)DNA
Plot
μL.
(B) Plot shows themagnetic
potential
changes
over
thevolumes
volumeofof
range
of0;5;0–50
μL
for
the
μL.
(B)
Plot
shows
the
potential
changes
over
the
volume
range
of
0–50
μL
for
the
DNA
shows
the potential changes
over the
volume
range
of 0–50
µL forone
the DNA
nanostructure-modified
nanostructure-modified
magnetic
beads.
Error
bars
represent
standard
deviation for three
nanostructure-modified
magnetic
Error bars
represent
one measurements.
standard deviation for three
magnetic
beads. Error bars
representbeads.
one standard
deviation
for three
measurements.
measurements.
3.4. The Detection of V. alginolyticus via Chronopotentiometry
Chronopotentiometry
3.4. The Detection of V. alginolyticus via Chronopotentiometry
Under the
theoptimal
optimalexperimental
experimental
conditions,
potentiometric
responses
V. alginolyticus
conditions,
thethe
potentiometric
responses
to V.to
alginolyticus
were
Under
the
optimal
experimental
conditions,
the
potentiometric
responses
to
V.
alginolyticus
were by
tested
using the polycation-sensitive
solid-contact polycation-sensitive
membrane
electrode
via
tested
using by
the solid-contact
membrane electrode
via chronopotentiometry.
were
tested
by using
thepotential
electrode
via
chronopotentiometry.
As
insolid-contact
Figureresponse
5A, thepolycation-sensitive
potential
responseincreases
V.membrane
alginolyticus
increases with
As
shown
in Figure
5A,shown
the
V. alginolyticus
with increasing
the
chronopotentiometry.
As
shown
in
Figure
5A,
the
potential
response
V.
alginolyticus
increases
with
increasing the of
concentration
of V.
alginolyticus,
indicates
that the
of the DNA
concentration
V. alginolyticus,
which
indicateswhich
that the
disassembly
of disassembly
the DNA nanostructures
increasing
the
concentration
of
V.
alginolyticus,
which
indicates
that
the
disassembly
of magnetic
the of
DNA
nanostructures
results in
inthe
thecharge
decrease
in theconcentration
charge or DNA
concentration
onmodified
the surface
the
results
in the decrease
or DNA
on the
surface of the
nanostructures
results
in
the
decrease
in
the
charge
or
DNA
concentration
on
the
surface
of
the
modified
magnetic
beads.
5B shows
calibration
curve ofaptasensing
the potentiometric
beads.
Figure
5B shows
theFigure
calibration
curvethe
of the
potentiometric
assay. It aptasensing
can be seen
modified
magnetic
beads.
5B shows
curve
of theis potentiometric
−1 ,assay
assay.
can
be
seen
the
linear
range the
for calibration
the aptasensing
proposed
potentiometric
is
that
theItlinear
range
forthat
theFigure
proposed
potentiometric
assay
10–100aptasensing
CFU mLaptasensing
and the
assay.
It
can
be
seen
that
the
linear
range
for
the
proposed
potentiometric
aptasensing
assay
is
−1
−1
−
1
10–100 CFU
mLis 10
, and
themL
detection
limit is 10 CFU mL .
detection
limit
CFU
.
10–100 CFU mL−1, and the detection limit is 10 CFU mL−1.
Figure 5.
5. (A)
(A) Potentiometric
Potentiometric responses
responses of
of the
the solid-contact
solid-contact polycation-sensitive
polycation-sensitive membrane
membrane electrode
electrode
Figure
Figure
5.
responses
of the
solid-contact
membrane
electrode
−1
DNA
in
1.5 mL
mL(A)of
ofPotentiometric
0.01 M
M sodium
sodium
chloride
solution
with 55polycation-sensitive
μg·mL
in
1.5
0.01
chloride
solution
with
µg
·mL−1 protamine
protamine and
and 40
40 μL
µL DNA
−1 protamine and 40 μL DNA
in
1.5
mL
of
0.01
M
sodium
chloride
solution
with
5
μg·mL
−
1
nanostructure-modified
magnetic
beads
in
the
presence
of
(a)
100;
(b)
50;
(c)
10;
and
(d)
0
CFU
mL−1
nanostructure-modified magnetic beads in the presence of (a–d) 100; 50; 10; 0 CFU mL V. alginolyticus.
nanostructure-modified
magnetic
in the presence
ofthe
(a) V.
100;
(b) 50; (c) 10;
and (d) 0 CFU
mL−1
1.
V.
alginolyticus.
(B)potential
Plot shows
the beads
potential
over
alginolyticus
concentration
range
(B)
Plot shows the
changes
over thechanges
V. alginolyticus
concentration
range
of 5–500 CFU
mL−of
V.
alginolyticus.
(B)
Plot
shows
the
potential
changes
over
the
V.
alginolyticus
concentration
range
of
−1
represent
one standard
for three measurements.
5–500 bars
CFUrepresent
mL . Error
Error
onebars
standard
deviation
for threedeviation
measurements.
5–500 CFU mL−1. Error bars represent one standard deviation for three measurements.
3.5. Specificity Test
3.5. Specificity Test
In order to test the specificity of the potentiometric aptasensing method for the detection of
In order to four
test the
specificity
of including
the potentiometric
aptasensing
for the detection
of
V. alginolyticus,
bacterial
strains
V. alginolyticus,
E. coli,method
Staphylococcus
aureus, and
V. alginolyticus,
four were
bacterial
strains
including
V. alginolyticus,
E. coli,
Staphylococcus
aureus,
and
Aeromonas
hydrophila
tested.
As shown
in Figure
6A, no obvious
potential
changes are
observed
Aeromonas
hydrophila
were
tested.
As
shown
in
Figure
6A,
no
obvious
potential
changes
are
observed
for these bacteria. This indicates that the proposed method for the detection of V. alginolyticus has a
for these bacteria. This indicates that the proposed method for the detection of V. alginolyticus has a
Sensors 2016, 16, 2052
7 of 9
3.5. Specificity Test
In order to test the specificity of the potentiometric aptasensing method for the detection of
V. alginolyticus, four bacterial strains including V. alginolyticus, E. coli, Staphylococcus aureus, and
Aeromonas
were tested. As shown in Figure 6A, no obvious potential changes are observed
Sensors 2016, hydrophila
16, 2052
7 of 9
for these bacteria. This indicates that the proposed method for the detection of V. alginolyticus has a
good
good specificity.
specificity. Additionally,
Additionally,the
theinfluence
influence of
ofthe
therandom
random DNA
DNA on
on the
the potential
potential response
response was
was also
also
investigated
investigated (Figure
(Figure 6B).
6B). The
The results
results indicate
indicate that
that the
the DNA
DNA nanostructures
nanostructures containing
containing the
the random
random
DNA
DNA would
would not
not induce
induceany
anysignificant
significantchange
changein
inthe
thepotential
potentialresponse.
response.
Figure 6.6. (A)
Potential changes
changes in
in the
the presence
presence of
of 500
500 CFU
CFU mL
mL−−11 Aeromonas
Aeromonas hydrophila
hydrophila (AH),
(AH),
Figure
(A) Potential
−1 V. alginolyticus. (B) Potential responses to 100
Staphylococcus
aureus
(SA),
E.
coli,
and
50
CFU
mL
−
1
Staphylococcus aureus (SA), E. coli, and 50 CFU mL
V. alginolyticus. (B) Potential responses to
−1 V. alginolyticus using the aptamer and random DNA-based DNA nanostructures. Error
CFU
mLmL
−1 V. alginolyticus using the aptamer and random DNA-based DNA nanostructures. Error
100
CFU
bars represent
representone
onestandard
standarddeviation
deviationfor
forthree
threemeasurements.
measurements.
bars
4. Conclusions
4. Conclusions
This work demonstrates a potentiometric aptasensing assay using signal amplification and
This work demonstrates a potentiometric aptasensing assay using signal amplification and
magnetic separation strategies. The method is based on the DNA nanostructure-modified magnetic
magnetic separation strategies. The method is based on the DNA nanostructure-modified
beads and the solid-contact polycation-sensitive membrane electrode for the reversible
magnetic beads and the solid-contact polycation-sensitive membrane electrode for the reversible
chronopotentiometric detection of V. alginolyticus. The proposed method shows a linear
chronopotentiometric detection of V. alginolyticus. The proposed method shows a linear concentration
concentration range of 10–100
CFU mL−1 with a detection limit of 10 CFU mL−1, and a good
range of 10–100 CFU mL−1 with a detection limit of 10 CFU mL−1 , and a good specificity for
specificity for the detection of V. alginolyticus. The proposed strategy can be used for the detection of
the detection of V. alginolyticus. The proposed strategy can be used for the detection of other
other microorganisms by changing the aptamers in the DNA nanostructures.
microorganisms by changing the aptamers in the DNA nanostructures.
Acknowledgments: This
This work
work was
was financially
financially supported
supported by
by the
the Strategic
Strategic Priority
Priority Research
Research Program
Program of
of the
the
Acknowledgments:
Chinese
Academy
of
Sciences
(XDA11020702),
the
National
Natural
Science
Foundation
of
China
(41176081,
Chinese Academy of Sciences (XDA11020702), the National Natural Science Foundation of China (41176081,
21475148
21475148 and
and 21575158),
21575158),and
andthe
theTaishan
TaishanScholar
ScholarProgram
ProgramofofShandong
ShandongProvince.
Province.
Author
Author Contributions:
Contributions: Jiawang
Jiawang Ding
Ding and
and Wei
WeiQin
Qinconceived
conceivedand
anddesigned
designed the
theexperiments;
experiments; Guangtao
Guangtao Zhao
Zhao
and Han Yu performed the experiments; Jiawang Ding and Guangtao Zhao analyzed the data, Guangtao Zhao,
and Han Yu performed the experiments; Jiawang Ding and Guangtao Zhao analyzed the data, Guangtao Zhao,
Tanji Yin, and Wei Qin wrote the paper.
Tanji Yin, and Wei Qin wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Conflicts of Interest: The authors declare no conflict of interest.
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