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Biosensors and Bioelectronics
journal homepage: www.elsevier.com/locate/bios
Nuclease-free target recycling signal amplification for ultrasensitive
multiplexing DNA biosensing
Zhihan Zhao
a,b
, Shixing Chen
b
, Jianbang Wang
b
, Jing Su
b
, Jiaqiang Xu
a,
⁎
, Sanjay Mathur
c
,
Chunhai Fan
b
, Shiping Song
b,
⁎
a
NEST Lab, Department of Chemistry, Shanghai University, Shanghai 200444, China
b
Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of
Sciences, 2019 Jialuo Road, Shanghai 201800, China
c
Institute of Inorganic Chemistry, University of Cologne, D-50939 Cologne, Germany
ARTICLE INFO
Keywords:
DNA detection
Nuclease-free
Target recycling
Multiplexing biosensing
ABSTRACT
Ultrasensitive biosensing technologies without gene amplification held great promise for direct detection of
DNA. Herein we report a novel biosensing method, combining target recycling signal-amplification strategy and
a homemade electrochemical device. Especially, the target recycling was achieved by a strand displacement
process, no needing the help of any nucleases. In the presence of target DNA, the recycling system could be
activated to generate a cascade of assembly steps with three hairpin DNA segments. Each recycling process were
accompanied by a disassembly step that the last hairpin DNA segment displaces target DNA from the complex
at the end of each circulation, freeing targets to activate the self-assembly of more trefoil DNA structures. This
biosensing method could detect target DNA at aM level and can distinguish target DNA from interfering DNAs,
demonstrating its high sensitivity and high selectivity. Importantly, the biosensing method could work well with
serum samples.
1. Introduction
The detection of genomic DNA of specific sequences is essential for
most biological assays, such as clinical diagnostics, functional geno-
mics, forensic analysis and even food safety (Chen et al., 2015; Kim and
Easley, 2011; Lubin and Plaxco, 2010; Wang et al., 2011). Currently,
most of the practical methods or kits for DNA detection need gene
amplification technologies such as polymerase chain reaction (PCR) or
isothermal amplification (Notomi et al., 2015). However, gene ampli-
fication processes often cause false positive due to the contaminated
samples, primer nonspecific binding or polymerase-caused errors (Sun
et al., 2016). Thus, ultrasensitive biosensing technologies without gene
amplification held great promise for direct detection of DNA (Gao and
Li, 2013).
Two strategies have attracted much attention to develop biosensing
technologies or methods for direct detection of DNA. One was the post-
amplification strategy towards the signal produced by hybridization or
recognition events. Even though high-sensitivity capability could be
achieved, this strategy still suffered from poor signal-to-noise ratio,
because background signals were often amplified if there was any
nonspecific binding of signaling probes. The other was target recycling
amplification strategy (Liu et al., 2013). The recycling amplification
process occurred only in the present of DNA targets. Thus it held great
promise for developing ultrasensitive DNA-sensing technologies.
However, nucleases such as endonuclease (Hun et al., 2010), exonu-
clease (Bi et al., 2012), polymerase (Song et al., 2011) or DNAzyme
(Zhao et al., 2013), were often involved in such strategy. These
nucleases usually costed high and needed special reaction condition
limiting their wide application.
With the development of DNA nanotechnology, hairpin assembly
amplification based on strand displacement has been reported for the
detection of DNA (Zhong et al., 2016), microRNA (Shi et al., 2016),
amyloid-beta oligomers (Zhu et al., 2016) and so on. In this work, we
designed a novel nuclease-free target recycling amplification strategy.
Target recycling was achieved only based on strand displacement
process without the help of nucleases. Combining the signal amplifica-
tion with a homemade multiplexing electrochemical biosensing device,
we developed an ultrasensitive method to directly detect DNA targets.
Compared with the limit of detection (LOD) for two hairpins displace-
ment in previous studies, e.g., 20 pM (Zhong et al., 2016), the LOD of
our method was 10.9 aM. Also, we demonstrated that the multiplexing
biosensing method worked well with biological fluids.
http://dx.doi.org/10.1016/j.bios.2017.03.051
Received 8 October 2016; Received in revised form 17 February 2017; Accepted 23 March 2017
⁎
Corresponding authors.
E-mail addresses: xujiaqiang@shu.edu.cn (J. Xu), spsong@sinap.ac.cn (S. Song).
Biosensors and Bioelectronics 94 (2017) 605–608
Available online 25 March 2017
0956-5663/ © 2017 Elsevier B.V. All rights reserved.
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