A label free and enzyme-free aptasensor for visual cd2+ detection based on split dn azyme fragments

This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
Accepted Manuscript
rsc.li/methods
Analytical
Methods
www.rsc.org/methods
ISSN 1759-9660
PAPER
Tetsuo Okada et al.
Chiral resolution with frozen aqueous amino acids
Volume 8 Number 1 7 January 2016 Pages 1–224
Analytical
Methods
View Article Online
View Journal
This article can be cited before page numbers have been issued, to do this please use: D. Zhou, W. Wu,
Q. Li, J. Pan and J. Chen, Anal. Methods, 2019, DOI: 10.1039/C9AY00822E.

Journal Name
ARTICLE
This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins
a.Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control
and Management, Guangdong Institute of Eco-environmental Science &
Technology, Guangzhou 510650, China.
E-mail: 222chenjunhua@163.com
b.College of Food Science and Engineering, Qingdao Agricultural University, No.700
Changcheng Road, Qingdao, 266109, China.
†Electronic Supplementary Information (ESI) available: [Supplementary Figures
(Fig. S1-S3)]. See DOI: 10.1039/x0xx00000x
‡The two authors contributed equally to this work.
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A label-free and enzyme-free aptasensor for visual Cd2+ detection
based on split DNAzyme fragments
Danhua Zhou,‡a Wei Wu,‡b Qiong Li,a Jiafeng Pan,a and Junhua Chena*
A label-free and enzyme-free three-way junction sensing system has been proposed for the amplified detection of Cd2+
based on the split G-quadruplex DNAzyme fragments. Three hairpin structures were designed as the building blocks
(hairpins 1, 2, and 3) to construct the three-way G-quadruplex junction. In the presence of Cd2+, the combination of Cd2+
and the aptamer initiated branch migration of hairpins to form an unstable complex. Then Cd2+- aptamer dissociated from
the complex, acting as a catalyst to trigger the hybridization of additional branched junctions. Thus, the 3:1 split G-
quadruplex fragments of those hairpins were brought into close-enough proximity to form G-quadruplex. After addition of
the hemin, numerous intact G-quadruplex/hemin DNAzymes were generated, leading to an intensified color change
observed by naked-eye. The biosensor exhibits excellent selectivity and high sensitivity for Cd2+ with a detection limit as
low as 10 pM. The biosensor is simple and convenient in operation without labeling, washing, and modification, which
makes it suitable for routine monitoring of Cd2+.
Introduction
As an extremely toxic heavy metal, cadmium could
cause severe damage to food safety and human health.1,2
The standard for Cd2+ in the drinking water was
recommended to be 5 ppb by The U.S. Environmental
Protection Agency (EPA).3 Due to its serious effects, it
caused extensive research to develop a sensitive and
reliable technique for Cd2+ detection. Several classical Cd2+
detection methods have been proposed, including
inductively coupled plasma mass spectrometry (ICP-MS),4
atomic absorption spectroscopy (AAS),5 and atomic
fluorescence spectroscopy (AFS),6 which offer high
sensitivity and accuracy. However, these methods usually
rely on complicated sample pre-treatment steps and
specialized instruments. As an alternative, colorimetric
biosensors possess many attractive advantages, such as
naked-eye readout, instrument-free format, short assay
time, and ease of use. These make the colorimetric
approach more suitable for the development of a rapid Cd2+
detection. But the colorimetric approach often suffers from
unsatisfactory sensitivity. Thus, it is quite essential to
develop a colorimetric sensing system for Cd2+ detection
with high sensitivity and simplified operation.
An essential and efficient way to construct sensing
systems with high sensitivity is adopting a signal
amplification strategy. In recent years, it has been reported
that various signal amplification strategies have been
achieved, including ligase chain reaction (LCR)7, rolling circle
amplification (RCA)8, and polymerase chain reaction (PCR),9
which require special reaction conditions and high cost.
Toehold-mediated DNA strand displacement reaction is a
better way to amplify signal comparing with those
methods. Toehold-mediated DNA strand displacement
reaction is a controllable non-enzymatic process in which
one strand of DNA in a double-stranded complex is
displaced by another DNA strand with the help of a short
over hanging single-stranded domain.10,11 This method
exhibits some intriguing characteristics, such as continuous
signal turnover capability, inherent modularity,
environmental robustness, and easy to scale up,12-14 which
make it more suitable for exquisitely sensitive molecular
detection.
Aptamers are one of the most promising nucleic acids
tool in the field of analytical chemistry. The essence of an
aptamer molecule is its three dimensional structure arising
from a folded single-stranded nucleic acid (usually 20-60
bases).15 Wu and co-workers16 chose Cd2+ as a model of a
small molecule with less sites, and proposed a novel SELEX
strategy of immobilizing ssDNA libraries on a matrix, for
selection of aptamers with high affinity to Cd2+. The
dissociation constant (Kd) of the selected aptamer was
determined as 34.5 nM for Cd2+,16 which demonstrated that
the binding affinity of the aptamer to Cd2+ is high. The
aptamer folds into a stem-loop structure when binding with
Page 1 of 7 Analytical Methods
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AnalyticalMethodsAcceptedManuscript
Publishedon17June2019.DownloadedbyAmericanUniversityofBeiruton6/19/20197:02:11PM.
View Article Online
DOI: 10.1039/C9AY00822E

ARTICLE Journal Name
2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx
Cd2+.16 This conformation change can be used for the design
of different aptasensors.
The G-quadruplex is a unique higher-order structure
consisting of square-planar arrangements of guanine
nucleobases stabilized by Hoogsteen hydrogen bonding and
monovalent cations.17 As an excellent signal reporter, it is
formed by G-rich sequences18 generally stabilized by alkali
metal cations, especially K+.19,20 G-quadruplex is
polymorphic, which can be used to design biosensors21,22
and DNA-based architectures.23-25 The G-quadruplex
sequence may be split into two fragments, which lack
catalytic activity. When the G-quadruplex is reformed from
the split fragments, the peroxidation activity is
reactivated.26-28 It is proven that in the presence of the
DNAzymes, H2O2-mediated oxidation of 3,3',5,5'-
tetramethylbenzidine (TMB) could be sharply accelerated
compared to the conditions without DNAzymes. And the G-
quadruplex was considered as a DNAzyme when interacted
with hemin, which can effectively catalyze the H2O2-
mediated oxidation of TMB to generate a color change.
Catalytic hairpin assembly (CHA) is a powerful signal
amplification tool for constructing various biosensors with
high sensitivity. For example, Yang and colleagues
developed an enzyme-free dual signal amplification
strategy for DNA detection based on CHA and split
DNAzyme fragments. In the presence of target DNA, the
active DNAzyme structures will be formed on each branch
end of the of three-arm DNA branched junctions, which can
catalyze the cleavage reaction toward the substrates to
generate the fluorescent signal.29 Wu group reported a
hairpin-type of both-end-blocked DNAzyme to improve the
signal-to-background ratio during the CHA process.30 The
both-end-blocked DNAzyme is by far the most sensitive
optical detection mode for monitoring the CHA process that
can be used for determination of 0.05 fmol miRNA-21.
In this study, we develop an enzyme-free and label-free
sensing platform for Cd2+ detection by integrating split G-
quadruplex DNAzyme with toehold-mediated strand
displacement and CHA. This sensing platform can amplify
the signal on the basis of a target induced three-way G-
quadruplex junction. Importantly, the output of the
colorimetric detector can be recognized readily by the
naked eye without requiring additional instrumentation.31-
34
Experimental
Materials and reagents
TMB, hemin, dimethyl sulfoxide (DMSO), Triton X-100,
hydrogen peroxide (H2O2, 30 wt.%), and tris-
(hydroxymethyl)aminomethane (Tris) were purchased from
Sigma-Aldrich (St. Louis, MO). Metal ions, including Hg2+,
Pb2+,Cd2+, Ag+, Zn2+, Mn2+, Ni2+, and Cu2+ were produced by
Sigma-Aldrich (USA). A hemin stock solution (5 mM) was
prepared in DMSO and stored in the dark at -20 °C. H2O2
was diluted to 3% using the buffer solution before use.
Other reagents and chemicals were of analytical grade and
used without purification. All solution was prepared with
ultrapure water (18.2 M/cm) from a Millipore Milli-
Qwater purification system (Billerica, MA).
All DNA oligonucleotides were ULTRAPAGE-purified
and purchased from Shanghai Sangon Biotechnology Co.,
Ltd. (Shanghai, China) and their sequences were listed as
follows:
aptamer: 5'-ATGGGTCTCACTATGGGACTGTTGTGGTATTATT
TTTGGTTGTGCAGTATGACTA-3'
DNA1: 5'-ATGGGTCTCACTATGGGATGACTA-3'
Hairpin1: 5'-TGGGTAGGGCGGGTTAGTCATCCCATAGTGAGA
CCCATATGGGTCAAGACATGGGTCTCACTATGGGT-3'
Hairpin2: 5'-TGGGTAGGGCGGGTAGTGAGACCCATGTCTTGA
CCCATATGGGATGACTAATGGGTCAAGACATGGGT-3'
Hairpin3: 5'-TGGGTAGGGCGGGTGTCTTGACCCATTAGTCAT
CCCATATGGGTCTCACTATGGGATGACTAATGGGT-3'
Assay procedure
The DNA solution was heated at 95 °C for 10 mins, and
gradually cooled to room temperature. Various
concentrations of Cd2+ was incubated with aptamer (100
nM), hairpin 1 (400 nM), hairpin 2 (400 nM), hairpin 3 (400
nM) in 20 mM Tris-HCl buffer (20 mM Tris, 50 mM NaCl, 25
mM KCl, 10 mM MgCl2, pH = 7.4) for 120 min at room
temperature to generate the three-way G-quadruplex
junction. Then 1 μM hemin was added to the resulting
product, and the mixture was incubated at room
temperature for 15 min to form the active hemin/G-
quadruplex DNAzymes. 50 μL of the final DNA-hemin
mixture solution was mixed with 150 μL of TMB-H2O2
substrate solution, which was constituted of 15 μL 0.5%
(w/v) TMB, 10 μL 3% (w/v) H2O2, and 125 μL substrate
buffer (containing 26.6 mM citric acid, 51.4 mM disodium
hydrogen phosphate, and 25 mM KCl, pH = 5.0). After
incubation at room temperature for 5 min, the colorimetric
responses of the sensor were observed by the naked eye
and the photos were taken by a digital camera. Meanwhile,
the absorption spectra of the solution were recorded by the
SpectraMax i3x (Molecular Devices) in the wavelength
range from 500 to 800 nm.
To investigate the specificity of the assay, other metal
ions including Hg2+, Pb2+, Ag+, Zn2+, Mn2+, Ni2+, and Cu2+at
100 nM were tested in the same way.
Result and discussion
The strategy for Hg2+ monitoring
The sensing principle of the platform for Cd2+ detection
is illustrated in Scheme 1. The aptamer, hairpin 1 (H1),
hairpin 2 (H2), hairpin 3 (H3) were used in this sensing
system. The two fragments of the split trigger DNA were
extended at the 5' and 3' ends of the aptamer (domains a
and b). Three hairpin structures were selected to construct
the three-way G-quadruplex junction. The two G-rich
Page 2 of 7Analytical Methods
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
View Article Online
DOI: 10.1039/C9AY00822E

Journal Name ARTICLE
fragments of the 3:1 split G-quadruplex sequences were
distributed at the 5' and 3' ends of those hairpins (domains
e and f). Three quarters of G-rich fragments were exposed
while one quarter of that functioned as part of stem of
hairpins. Those hairpins all included trigger DNA (domains
b*, d, and c). In the absence of Cd2+, the aptamer is
metastable. Domain a and domain b are separated, which
cannot catalyze the toehold-mediated strand displacement
among those hairpins (H1, H2, and H3) to initiate the
amplification reaction. Therefore, G-quadruplex DNAzyme
cannot be formed, resulting that no color signal could be
observed. With the addition of Cd2+, the Cd2+-aptamer
complex is formed, which brings domains a and b into
close-enough proximity to catalyze the toehold-mediated
strand displacement. Domain b first binds with the exposed
domain b* of H1, initiating branch migration to open H1.
The combination of H1 and the Cd2+-aptamer complex
results in the exposure of domain c*. Then domain c of H2
interacts with the newly open domain c* of H1 to mediate
branch migration again to open H2, which causes domain
d* to no longer be closed. Finally, domain d of H3 can
hybridize to the newly accessible domain d* of H2 to
activate toehold-mediated DNA strand displacement
reaction. H3 is open then displaces the Cd2+-aptamer
complex, allowing the Cd2+-aptamer complex to act as a
catalyst to trigger the hybridization of additional branched
junctions. H1, H2, and H3 form the complex (three-way G-
quadruplex junction), bringing the 3:1 split G-quadruplex
fragments into close-enough proximity and stimulate the
formation of intact G-quadruplex. After addition of the
hemin into the sensing system, G-quadruplex binds with the
hemin to form catalytic G-quadruplex/hemin peroxidase-
mimicking DNAzymes, which can effectively catalyze the
H2O2-mediated oxidation of TMB to generate a color signal
readout. The signal readout can be easily recognized by the
naked eye.
Scheme 1. Schematic illustration of the label-free DNA Y
junction for Cd2+ monitoring using G-quadruplex as signal
reporter with a naked-eye readout.
Feasibility study
To verify the feasibility of the strategy, the absorption
spectra and color changes of the solution at different
conditions were shown in Fig. 1. In the presence of
aptamer, H1, H2 and H3 but no Cd2+, there was no
significant color changed and the biosensor exhibited a
negligible response (curve a), which implied that the
hairpins could coexist in solution with negligible
intermolecular hybridization without Cd2+. G-quadruplex
DNAzyme cannot be formed, so color signal was not
significant. Upon addition of Cd2+, aptamer, and H1 to the
system, the solution showed colorless and the absorbance
(curve b) showed almost no change relative to curve a.
When both H1 and H2 were present in the mixture, the
absorbance intensity increased (curve c). And the color of
the solution turned blue. Because active DNAzyme was
generated in the presence of aptamer-H1-H2 compound.
Noted that, when Cd2+, aptamer, H1, H2, and H3 were
present in the sensing system, the absorbance intensity
dramatically increased (curve d). The reason is that our
rationally designed hairpins could induce the autonomous
cyclic formation of H1-H2-H3 product to generate G-
quadruplex DNAzyme. Upon the addition of Cd2+, the Cd2+-
aptamer complex is formed to open H1, H2, and H3. Then
the Cd2+-aptamer complex was displaced during toehold-
mediated DNA strand displacement reaction. Three-way G-
quadruplex junction was formed, thereby generating
numerous G-quadruplex DNAzyme molecules to give an
amplified colorimetric signal. In addition, the color of the
solution changes blue (inset in Fig. 1), which further
demonstrated the feasibility of our strategy. These results
clearly indicated that the proposed three-way G-quadruplex
junction sensing system can be applied for Cd2+ detection.
The Cd2+-aptamer containing domains a and b formed a
hairpin structure to open hairpin 1. The reaction rate
should be slower than the single stranded DNA containing a
and b domains (DNA1). In order to further prove that the
strategy is feasible, their reaction kinetics should be
compared. As shown in Fig. S1 (ESI†), the absorbance of the
sensing system containing Cd2+-aptamer increased along
with increasing of the reaction time, and kept almost a
constant level after 120 min (curve B). The absorbance of
the sensing system containing DNA1 also increased along
with increasing of the reaction time within 100 min, and
the reaction rate was much faster. After 100 min, the
reaction equilibrium was reached (curve A).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
View Article Online
DOI: 10.1039/C9AY00822E

Fig. 1. Feasibility of the label-free colorimetric sensing
platform. (a): aptamer (100 nM) + H1 (400 nM) + H2 (400
nM) + H3 (400 nM). (b): Cd2+ (100 nM) + aptamer (100 nM)
+ H1 (400 nM). (c): Cd2+ (100 nM) + aptamer (100 nM) + H1
(400 nM) + H2 (400 nM). (d): Cd2+ (100 nM) + aptamer (100
nM) + H1 (400 nM)+ H2 (400 nM)+ H3 (400 nM). The inset
showed different colors of these tested solution.
Experiments are performed in TMB-H2O2 reaction solution.
Optimization of assay conditions
The results of this experiment are affected by many
conditions such as the concentration of hairpins, the
reaction time, and the temperature. The experimental
conditions were optimized to achieve the best sensing
performance.
In this study, the concentration of hairpins plays a
critical role in the performance of the sensing system. The
effect of concentration of hairpins on the response of the
sensor was shown in Fig. S2 (ESI†). With increasing of the
hairpins concentration, the absorbance intensity of the
system containing 100 nM Cd2+ increased dramatically
within 400 nM. Then it has not changed significantly further
increasing the concentration of hairpins (yellow histogram).
However, with the increase of the hairpins concentration,
the absorbance intensity without target (the background
signal) was also enhanced slightly (pink histogram). Thus,
the signal-to-noise (S/N) ratio increased gradually. In view
of the highest net signal, the hairpins concentration should
be 400 nM for the subsequent experiment.
The stability of the hairpin structure was influenced by
the reaction temperature of the system. Therefore, the
effect of reaction temperature on the response of the
sensor was also investigated by detecting 100 nM Cd2+ at
different temperatures (4 °C, 25 °C, 37 °C, and 45 °C). The
blank sample (in the absence of Cd2+) at each temperature
was conducted at the same conditions. As shown in Fig. S3
(ESI†), in the presence of Cd2+, the absorbance intensity of
the system increased dramatically in the range from 4 to 37
°C and then leveled off to a saturation value when the
temperature was higher than 37 °C (blue histogram). On
the other hand, the absorbance intensity without target
(the background signal) was also enhanced (green
histogram). The S/N ratio was the best when the reaction
temperature was 25 °C. The reason might be attributed to
the fact that the structure of the hairpin probes could be
destroyed with increased temperature, which elevated the
background signal. It is more advisable that 25 °C was the
best reaction temperature.
The reaction time is also non-negligible factor in the
sensing system. The process of signal amplification was
strongly affected by the reaction time of the toehold-
mediated strand displacement. As displayed in Fig. S4
(ESI†), in the presence of Cd2+, the colorimetric signal
elevated gradually with the augment of reaction time
within 120 min. Then, it has not changed significantly
further increasing the reaction time (red curve and blue
curve), which indicated that the two G-rich fragments
almost formed into G-quadruplex DNAzyme. The
background signal (in the absence of Cd2+) was also
enhanced slightly along with increasing the reaction time
(black curve). To obtain the maximal signal amplification
efficiency, 120 min was selected as the optimum reaction
time.
Analytical performance of the biosensor
Under the optimal conditions, different concentrations
of Cd2+ were added to the reaction system to investigate
the dynamic range and sensitivity of the assay. As shown in
Fig. 2A, when Cd2+ is absent, no significant color change was
observed, that is, the solution was almost colorless. With
increasing Cd2+ concentration, the color of the solution
turned from colorless to deep blue gradually , indicating
that G-quadruplex DNAzyme were generated through
toehold-mediated DNA strand displacement reaction. The
absorbance spectra of the solution recorded by the
SpectraMax i3x further confirmed their corresponding
colorimetric responses. The absorbance enhanced gradually
along with the increase of the target concentration (Fig.
2B). A proportional linearity between the absorbance
spectra and the logarithm of Cd2+ concentration was
obtained in the range from 10 pM to 1 μM. The absorbance
values at 650 nm were also recorded in Fig. 2C. The
resulting calibration curve demonstrated that the
absorbance values are proportional to the logarithm of Cd2+
concentrations. The correlation equation can be expressed
as follow: y = 0.1291 lg C + 0.1732 (R2 = 0.98) (y and C
represent the absorbance value and the concentration of
Cd2+). According to the distinct color difference between
the sample and the blank test, the presence of Cd2+ higher
than 10 pM (including 10 pM Cd2+) can be easily identified
by the naked eye. Thus, the visual detection limit of our
Cd2+ biosensor is 10 pM. Compared with the previously
reported detection of Cd2+, the sensing system proposed
herein displays higher sensitivity, which could credit to the
continuous turnover capability of the toehold-mediated
strand displacement reaction. With the impressive visual
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
View Article Online
DOI: 10.1039/C9AY00822E

Journal Name ARTICLE
detection limit and simple operation, the strategy avoiding
the involvement of any DNA immobilization holds great
promise for Cd2+ monitoring with high sensitivity.
To investigate the selectivity of this method, several
other metal ions including Hg2+, Pb2+, Ag+, Zn2+, Mn2+, Ni2+,
and Cu2+ at the same concentration of 100 nM were also
performed by the present biosensor. As displayed in Fig. 3,
only the sample with Cd2+ revealed a clearly intensified
color change while the samples with other metal ions
displayed no obvious color change, demonstrating that our
sensing system achieve high selectivity. Such high selectivity
can be primarily attributed to the highly specific binding
capability of the Cd2+ to the aptamer.
Fig. 2. (A) The color change of the samples with different
Cd2+ concentration. (B) The absorbance spectra of the label-
free colorimetric sensing platform of various concentrations
of Cd2+. (C) Linear relationship between the absorbance
spectra and various concentrations of Cd2+. The error bars
denote the standard deviation of three independent
measurements.
Fig. 3. The color change of the samples with different metal
ions. The concentration of metal ions is 100 nM.
Real sample analysis
Real river water samples were tested to investigate the
practical application of the biosensor. Different
concentrations of Cd2+ (5 nM, 20 nM, 40 nM, 60 nM, and 80
nM) were added to the river water to detect the recovery.
The results were showed in Table 1. The recovery was in
the range from 96.5%-105.3%, which indicated that the
possible interference from the real water samples was
negligible.
Table 1. The recovery of different concentrations of Cd2+ in
river water using the proposed biosensor.
River water Added (nM) Founda (nM) Recovery (%)
1 5 5.2 104
2 20 21 105
3 40 38.6 96.5
4 60 63.2 105.3
5 80 79.1 98.9
aMean of three determinations.
Moreover, the Cd2+ concentrations in tap water, lake
water, and pond water were also validated by comparing to
the standard AFS (atomic fluorescence spectrometer)
analysis. The data in Table 2 revealed that no significant
difference existed between the values measured using the
proposed biosensor and the AFS method, confirming the
accuracy of the developed sensor for Cd2+ detection.
Table 2. Determination of Cd2+ in environmental water
samples using the proposed biosensor and AFS.
Samples AFSa (pM)
Proposed
methodb (pM)
Relative error
(Re) c (%)
Tap water 0.87 0.91 4.6
Lake water 4.67 4.96 6.2
Pond water 9.32 9.18 -1.5
aEach sample was analyzed using the standard AFS (atomic
fluorescence spectrometer), and all values were obtained
as an average of three repetitive determinations.
bEach sample was analyzed using our proposed biosensor,
and all values were obtained as an average of three
repetitive determinations.
cOur proposed method vs. AFS.
Conclusions
In conclusion, a colorimetric assay for the amplified
detection of Cd2+ was successfully developed on the basis of
a target-induced three-way G-quadruplex junction. The
working principle is based on toehold-mediated strand
displacement reactions, which can take place without any
enzyme. Three hairpin structures (H1, H2, and H3) were
selected to construct the three-way G-quadruplex junction
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
View Article Online
DOI: 10.1039/C9AY00822E

via a series of toehold-mediated strand displacement
reactions to amply cascaded signal. In the presence of Cd2+,
numerous intact G-quadruplex/hemin DNAzymes were
generated, leading to an intensified color change of the
reaction solution for visual detection of Cd2+ down to 10 pM
without instrumentation. The assay exhibits excellent
selectivity toward Cd2+ against other metal ions. This
method can be applied to the determination of Cd2+ in river
water samples with satisfactory recovery and accuracy.
Additionally, the measurement procedure is carried out
under isothermal conditions. The construction strategy
described herein is economic in operation without labelling,
washing, and modification, requiring only the mixing of
several solutions to achieve visible and intuitive results,
which makes it suitable for routine monitoring of Cd2+. With
the above characteristics, the developed biosensor can be
extended to be a universal sensing platform for other
targets detection by substituting the target-recognition
element.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Financial support was provided by the Key Projects in the
National Science & Technology Pillar Program of China
(Grant No. 2015BAD06B03).
Notes and references
1 J. Godt, F. Scheidig, C. Grosse-Siestrup, V. Esche, P.
Brandenburg, A. Reich and D.A. Groneberg, J. Occup.
Med. Toxicol., 2006, 1, 1-6
2 A. A. Meharg, G. Norton, C. Deacon, P. Williams, E. E.
Adomako, A. Price, Y. Zhu, G. Li, F. J. Zhao, S. McGrath,
A. Villada, A. Sommella, P. M. De Silva, H. Brammer, T.
Dasgupta and M. R. Islam, Environ. Sci. Technol., 2013,
47, 5613-5618.
3 L. Zeng, J. Gong, P. Rong, C. Liu and J. Chen, Talanta
2019, 198, 412-416.
4 C. D. Palmer, M. E. Lewis, C. M. Geraghty, F. Barbosa
and P. Parsons, J. Spectrochim. Acta, Part B, 2006, 61,
980-990.
5 C. P. P. Karapatouchas and A. N. Anthemidis,
Microchim. Acta 2008, 160, 455-460.
6 X. Guo and X. Guo, Anal. Chim. Acta 1995, 310, 377-
385.
7 M. Liu, W. Zhang, Q. Zhang, J. D. Brennan and Y. Li,
Angew. Chem. Int. Edit., 2015, 54, 9637-9641.
8 F. Wang, C. Lu, X. Liu, L. Freage and I. Willner, Anal.
Chem., 2014, 86, 1614-1621.
9 Y. Gao and B. Li, Anal. Chem., 2014, 86, 8881-8887.
10 R. Deng, L. Tang, Q. Tian, Y. Wang, L. Lin and J. Li,
Angew. Chem. Int. Ed., 2014, 53, 2389-2393.
11 J. Chen, S. Zhou and J. Wen, Anal. Chem., 2014, 86,
3108-3144.
12 F. Huang, H. Xu, W. Tan and H. Liang, ACS Nano 2014,
8, 6849-6855.
13 T. Song, S. Xiao, D. Yao, F. Huang, M. Hu and H. Liang,
Adv. Mater., 2014, 26, 6181-6185.
14 T. Song and H. Liang, J. Am. Chem. Soc., 2012, 134,
10803-10806.
15 M. Mascini, I. Palchetti and S. Tombelli, Angew. Chem.
Int. Ed., 2012, 51, 1316-1332.
16 Y. Wu, S. Zhan, L. Wang and P. Zhou, Analyst 2014,
139, 1550-1561.
17 X. Liu, M. Chen, T. Hou, X. Wang, S. Liu and F. Li,
Biosens. Bioelectron., 2014, 54, 598-602.
18 T. Li, E. Wang and S. Dong, Chem. Commun., 2009,
580-582.
19 J. T. Davis, Angew. Chem., Int. Ed., 2004, 43, 668-698.
20 J. F. Burnett and A. Walmsley, Biochem., 1999, 38,
14063-14068.
21 D. Hu, Z. Huang, F. Pu, J. Ren and X. Qu, Chem.-Eur. J.,
2011, 17, 1635-1641.
22 D. Miyoshi, A. Nakao and N. Sugimoto, Nucleic Acids
Res., 2003, 31, 1156-1163.
23 C. Wang, Z. Huang, Y. Lin, J. Ren and X. Qu, Adv.
Mater., 2010, 22, 2792-2798.
24 K. Dutta, T. Fujimoto, M. Inoue, D. Miyoshi and N.
Sugimoto, Chem. Commun., 2010, 46, 7772-7774.
25 S. Shimron, N. Magen, J. Elbaz and I. Willner, Chem.
Commun., 2011, 47, 8787-8789.
26 J. Zhu, L. Zhang, T. Li, S. Dong and E. Wang, Adv.
Mater., 2013, 25, 2440-2444.
27 J. Zhu, L. Zhang, S. Dong and E. Wang, ACS Nano 2013,
7, 10211-10217.
28 L. Hu, X. Liu, A. Cecconello and I. Willner, Nano Lett.,
2014, 14, 6030-6035.
29 K. Yang, M. Zeng, X. He, J. Li and D. He, Anal. Methods
2016, 8, 8262-8265.
30 L. Deng, Y. Wu, S. Xu, Y. Tang, X. Zhang and P. Wu, ACS
Sens., 2018, 3, 1190-1195.
31 S. Shimron, F. Wang, R. Orbach and I. Willner, Anal.
Chem., 2012, 84, 1042-1048.
32 J. Chen, S. Chen and F. Li, Chem. Commun., 2017, 53,
8743-8746.
33 J. Chen, S. Zhou and J. Wen, Angew. Chem. Int. Ed.,
2015, 54, 446-450.
34 X. Hu, C. Li, C. Feng, X. Mao, Y. Xiang and G. Li, Chem.
Commun., 2017, 53, 4692-4696.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
View Article Online
DOI: 10.1039/C9AY00822E

A label-free and enzyme-free three-way G-quadruplex junction sensing system was developed
for Cd2+ detection with a naked-eye readout.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
View Article Online
DOI: 10.1039/C9AY00822E

A label free and enzyme-free aptasensor for visual cd2+ detection based on split dn azyme fragments

Recommended

Recommended

More Related Content

Similar to A label free and enzyme-free aptasensor for visual cd2+ detection based on split dn azyme fragments

Similar to A label free and enzyme-free aptasensor for visual cd2+ detection based on split dn azyme fragments (20)

Recently uploaded

Recently uploaded (20)

A label free and enzyme-free aptasensor for visual cd2+ detection based on split dn azyme fragments