Design of nuclease-based target recycling signal amplification in aptasensors

Biosensors and Bioelectronics 77 (2016) 613–623

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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Design of nuclease-based target recycling signal amplification in aptasensors Mengmeng Yan a,b, Wenhui Bai a,b, Chao Zhu a,b, Yafei Huang a,b,c, Jiao Yan a,b,c, Ailiang Chen a,b,n a Institute of Quality Standards and Testing Technology for Agro-products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of Agricultural Science, Beijing 100081, China b Key Laboratory of Agri-Food Quality and Safety, Ministry of Agriculture, Beijing 100081, China c College of Food Science and Technology, Hainan University, Haikou 570228, China

art ic l e i nf o

a b s t r a c t

Article history: Received 11 August 2015 Received in revised form 21 September 2015 Accepted 5 October 2015 Available online 16 October 2015

Compared with conventional antibody-based immunoassay methods, aptasensors based on nucleic acid aptamer have made at least two significant breakthroughs. One is that aptamers are more easily used for developing various simple and rapid homogeneous detection methods by “sample in signal out” without multi-step washing. The other is that aptamers are more easily employed for developing highly sensitive detection methods by using various nucleic acid-based signal amplification approaches. As many substances playing regulatory roles in physiology or pathology exist at an extremely low concentration and many chemical contaminants occur in trace amounts in food or environment, aptasensors for signal amplification contribute greatly to detection of such targets. Among the signal amplification approaches in highly sensitive aptasensors, the nuclease-based target recycling signal amplification has recently become a research focus because it shows easy design, simple operation, and rapid reaction and can be easily developed for homogenous assay. In this review, we summarized recent advances in the development of various nuclease-based target recycling signal amplification with the aim to provide a general guide for the design of aptamer-based ultrasensitive biosensing assays. & 2015 Elsevier B.V. All rights reserved.

Keywords: Aptamer Aptasensors Nuclease Target recycling Signal amplification

Contents 1.

2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Aptamer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclease-based target recycling signal amplification aptasensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Exonuclease III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Fluorescent methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Electrochemical methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Chemiluminescence methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. Colorimetric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Nt.BbvCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Fluorescence methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Colorimetric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. RecJf exonuclease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Electrochemical method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Electrochemiluminescence method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. DNase I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

614 614 614 615 615 615 617 618 618 618 618 619 619 619 619 619 619

n Corresponding author at: Institute of Quality Standards and Testing Technology for Agro-Products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of Agricultural Sciences, Beijing 100081, China. Fax: þ86 10 82106560. E-mail address: [email protected] (A. Chen).

http://dx.doi.org/10.1016/j.bios.2015.10.015 0956-5663/& 2015 Elsevier B.V. All rights reserved.

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2.4.1. Fluorescence methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619 2.4.2. Electrochemical methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620 2.5. Other nucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620 2.5.1. Exonuclease I (Exo I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620 2.5.2. T7 exonuclease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620 2.5.3. S1 nuclease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620 2.5.4. Nt.AlwI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621 3. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621 4. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622 Appendix A. Supplementary material. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622

1. Introduction 1.1. Aptamer Aptamers, also known as chemical antibodies, are singlestranded DNA or RNA that can bind to a wide range of molecules with high specificity and affinity (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Their lengths usually vary from 10 to 80 bases selected from a combination DNA library by in vitro selection termed Systematic Evolution of Ligands EXponential enrichment (SELEX) (Gopinath, 2007; Zhu et al., 2014). Similar to antibodies, aptamers can adopt specific three-dimensional conformations to combine with target analytes including stems, internal loops, purine-rich bulges, hairpin structures, hairpins, pseudoknots, kissing complexes, and G-quadruplex structures. With similar affinity to substances at concentrations from nM to pM, aptamers show more advantages than conventional antibodies. Among the advantages, the most important three are: (1) Target diversity: antibodies are prepared by animal immunization in physiological conditions, and many toxic chemicals or non-immunogenic small molecules are difficult to obtain for preparing antibodies. But for aptamers prepared in vitro by SELEX, nearly all chemicals could be used as the target for aptamer selection under any favorable conditions. To date, various aptamers have been identified for hundreds of targets ranging from small ions (e.g. Zn2 þ (Ciesiolka et al., 1995) and K þ (Qin et al., 2010)) and small organic compounds (e.g. organic dyes (Wilson and Szostak, 1998), neutral disaccharides (Yang et al., 1998), and antibiotics (Kwon et al., 2014; Song et al., 2012b)) to large molecules like glycoproteins (such as CD4 (Zhao et al., 2014)) or even more complex targets (e.g. whole living cells (Cerchia et al., 2009)). (2) High stability: as oligonucleic acid of 10–80 bases, aptamer shows high thermal stability and can be stored and transported at room temperature and easily refolded after denaturation induced by heat or other conditions. Furthermore, the stability is also reflected in its complete batch-to-batch consistency since aptamer is produced by chemical synthesis upon acquirement of the sequences. The aptamer stability is of great importance in reliability of aptasensor results. (3) Low cost: unlike antibodies whose production relies on animal immunization or cell culture, aptamers are easily prepared in large scale by simple chemical synthesis with very cheap nucleotides. High thermal stability also reduces the storage and transportation cost. Thus, aptamers are superior to antibodies in commercial assay kit development for biomedical or food safety analysis. Because of these predominant advantages, aptamers have received significant attention, and various aptamer-based bioassays including fluorescent, colorimetric, and electrochemical methods have been developed and extensively adopted for an impressively wide variety of applications in clinical, medical, environmental

monitoring, and food analysis (Dong et al., 2014; Santosh and Yadava, 2014; Wang and Farokhzad, 2014; Wu et al., 2014; Yoshida et al., 2014). 1.2. Aptasensor As we all know, simplicity and sensitivity are always two goals for analytical scientists. Antibody-based immunoassays have been developed for many years. Two commonly used methods ELISA and LFIA require multi-step washing and are low sensitive. Now, aptamer-based sensors called aptasensors have made significant breakthroughs in the development of homogeneous and highly sensitive assays. Unlike immunoassays in which antibodies or antigens are always immobilized on solid substrates including microplate wells or nitrocellulose membrane, aptamers are seldom used in such a solid–liquid sensing system even though they have the ability to develop similar sensing approaches such as various ELISA (Ikebukuro et al., 2005; Park et al., 2014; Ruslinda et al., 2012; Tennico et al., 2010; Toh et al., 2015), SPR (Bai et al., 2013b), quartz crystal microbalance (Tombelli et al., 2005), cantilever (Bai et al., 2014; Lim et al., 2014), or field-effect transistors (Martinez et al., 2009). As nucleic acid-based molecular recognition elements, aptamers show more flexibility in designing homogeneous and sensitive sensing formats by using intra- and inter-molecular hybridization, enzymatic replication, as well as easy sequence determination characteristics (Guo et al., 2014; Lau and Li, 2014; Li et al., 2012, 2014; Sassolas et al., 2011b; Sosic et al., 2013; Wu et al., 2015b; Zhang et al., 2014d; Zhou et al., 2014). The homogeneous aptasensor design has been reviewed by Li et al. (2010), Sassolas et al. (2011a), and Zhang et al. (2013a). But so far, there are no comprehensive reviews about highly sensitive aptasensor designs with various signal amplification methods. Target detection has been developed based on recognition between target molecules and aptamers in a 1:1 stoichiometric ratio. The stoichiometric recognition, however, puts an intrinsic limitation on detection sensitivity because one target molecule produces only one signal probe. To break through the limitation and achieve high sensitivity, three technologies for signal amplification have been reported. (1) The first signal amplification strategy involves a single signal-reporting tag that is able to incorporate numerous detectable elements. The strategy includes various mimicking enzyme catalysis (Chen et al., 2015a; Cheng et al., 2014; Gui et al., 2014; Liu et al., 2014a; Nie et al., 2013; Xia et al., 2015; Yuan et al., 2014a, 2014b; Zhang et al., 2014b; Zhao et al., 2015; Zheng et al., 2015; Zhuo et al., 2014), metal nanoparticles such as AuNPs (Fang et al., 2015; Liu et al., 2015; Song et al., 2014), silver enhancement (Chen et al., 2014; Ocana and del Valle, 2014; Shan et al., 2014), nanomaterials carrying multivalent signal probes (Chen et al., 2015b; Liu et al., 2014b; Yang et al., 2014c; Zhao et al., 2012a), QDs (Sheng et al., 2012), and bio-barcoded (Xia et al., 2015; Zhang et al.,

M. Yan et al. / Biosensors and Bioelectronics 77 (2016) 613–623

2009b), electrochemical or ECL active compounds (Huang et al., 2015a; Jiang et al., 2012; Li et al., 2015; Su et al., 2015; Wang et al., 2013; Wu et al., 2015d, 2013a; Zhang et al., 2014c), and two of them are often combined in one assay (Xia et al., 2015; Zhang et al., 2013b). These techniques are very important, based on which signal amplification approaches require tedious label preparation and conjugation procedures. In addition, a 1:1 stoichiometric ratio is usually used on the recognition interaction between the target and the signal-reporting unit in these technologies. An inherent limitation in the recognition interaction lies in detection sensitivity due to the generation of only one signal probe for detection by each target protein molecule. (2) The second signal amplification strategy involves nucleic acid amplification in which oligonucleotide sequences are used as signal units. This amplification could be divided into polymerase-dependent and polymerase-independent classes. The former includes PCR (Song et al., 2012a; Zhang et al., 2006), RCA (Liang et al., 2014; Shen et al., 2015; Wu et al., 2015c, 2010), real-time PCR (Lee et al., 2009), RNA polymerase-based amplification (Zhao et al., 2012b), PLA (Guthrie et al., 2006), and HCR (Peng et al., 2014b; Zheng et al., 2014), and these approaches are often combined with the first signal amplification approaches in an assay (Tang et al., 2012b). The latter includes HCR (Chen et al., 2015a; Song et al., 2015; Wang et al., 2015; Wu et al., 2015a), strand displacement-driven assembly of multiple G-quadruplex DNAs (Xu et al., 2015), and DNA concatemers (Bai et al., 2013a; Jiang et al., 2013c). These techniques are highly sensitive in detection, but these sensing protocols are limited due to complex handling operations, easy contamination and high cost. Therefore, search for novel strategies of simple and sensitive aptasensors remains a challenge. (3) Recently, nuclease-based signal amplification with aptamer-based sensing approaches has received wide attention largely because of easy homogeneous detection, high sensitivity, and a potential for high-throughput analysis. By using a variety of endonucleases, exonucleases, and nucleases, the target recycling amplification systems in various states for increasing the signal/target ratio have been designed including electrochemistry (Cao et al., 2012; Liu et al., 2013b), fluorescence (Lu et al., 2010b; Xue et al., 2012), chemiluminescence (Bi et al., 2014), and colorimetric (Li et al., 2012) approaches. Table 1 describes the commonly used nucleases in aptasensors with signal amplification. In this review, we summarized recent advances in the development of various nuclease-based target recycling signal amplification approaches with the aim to provide a general guide for the design of aptamer-based ultrasensitive biosensing assays. Nevertheless, some gaudy but complicated and unpractical systems are referred to but not detailed in this review. Table 2 lists the recent

615

nuclease-based signal amplification aptasensors in terms of the target, nuclease, method and sensitivity.

2. Nuclease-based target recycling signal amplification aptasensor 2.1. Exonuclease III Exo III is a 3′–5′ exonuclease enzyme and catalyzes stepwise removal of mononucleotides from the 3′-hydroxyl terminus of double-stranded DNA while the preferred substrates are blunt or recessed 3′-termini (Mol et al., 1995). The enzyme is not active on ssDNA or duplex DNA with a protruding 3′-end (Zuo et al., 2010b). The degree of resistance depends on the extension length, with the extensions of 4 bases or longer being essentially resistant to cleavage. Due to its accessibility, high efficiency and sequenceindependence, Exo III provides a more versatile platform for biomolecule detection and is first reported as an effective catalytic label for amplified detection of DNA (Freeman et al., 2011; Lee et al., 2005; Zhang et al., 2011; Zhao et al., 2011; Zuo et al., 2010a). With the nucleic acid characteristics of aptamer, Exo III has been introduced into aptamer-based sensors, in which analytes could be regenerated by the Exo III activity, resulting in repeated recycling of recognition events and formation of readout signals. 2.1.1. Fluorescent methods Aptamers have known sequences and are easily labeled with various fluorophores during chemical synthesis. Therefore, fluorescence-based detection is one of the most commonly used methods for designing aptamer-based assays due to its simplicity and sensitivity. Various aptamer-based MBs have been designed to detect a wide range of ligands (Giannetti et al., 2013; Hamaguchi et al., 2001; Zhang et al., 2014a). To increase the sensitivity of Exo III, Liu et al. (2012b) elaborately designed a nucleic acid hairpin modified with a 6-carboxyfluorescein (FAM) dye as a fluorophore at its 5′ terminus and a BHQ1 at its 3′ terminus. An aptamer sequence for ATP that is confined in the stem′s duplex structure is included in the hairpin structure. As shown in Fig. S1A (Supplemental information), nucleic acid self-hybridizes without ATP to form a hairpin structure including an Exo III-resistant 3′ terminus, which is a single strand. This draws the fluorophore and quencher close so that the fluorophore is quenched and a low fluorescence signal is resulted. In contrast, the hairpin is reconfigured with ATP to form an active G-quadruplex structure that can be bound to ATP. After the G-quadruplex hairpin structure is reorganized, the 3′end nucleic acid that carries BHQ1 is combined with the stem

Table 1 Commonly used nucleases in aptasensors with signal amplification. Type

Nuclease

Specific recognition

Substrate

Endonuclease

Nt.BbvCI Nt.AlwI S1 nuclease

þ þ 

One stand of DNA on dsDNA – One stand of DNA on dsDNA ssDNA/ssRNA single-stranded – regions in duplex DNA

– – –

Exonuclease

Exo III



dsDNA

3′–5′

Exo I T7 Exo RecJf

  

ssDNA One stand of DNA on dsDNA ssDNA

3′–5′ 5′–3′ 5′–3′

3′-Hydroxyl terminus with blunt or recessed extensions of 4 bases or longer being essentially resistant to cleavage. – 5′-Hydroxy termini of duplex DNAs –



ssDNA/dsDNA

–

–

Deoxyribonuclease DNase I

Degradation direction

Terminal limit

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Table 2 Recent nuclease-based signal amplification aptasensors. Enzyme

Target

Method

Detection Limit

Ref.

Exonuclease III

ATP Cocaine TB ATP DNA ATP TB Adenosine TB ATP DNA TB TB ATP TB Adenosine IFN-λ PDGF-BB Adenosine

Fluorescence

0.25 μM 50 nM 89 pM 9.5 nM 5 nM 10 μM 1 nM 1 nM 0.2 pM 1 nM 0.1 nM 5 pM 45 fM 34 pM 2 fM 0.5 nM 0.1 pM 0.68 nM 5.6 nM

Liu et al. (2012b)

100 pM 5 pM 19 pM 2.0 pM 1 fM 1 pM 6 pM 50 pM 40 cells 1.5 pM 3.0  10  13 g/mL 80 fM 8.1 pM 6.9 pM 1.0 fM

Xue et al. (2012) Feng et al. (2012) Li et al. (2014) Huang et al. (2015b)

Nt.BbvCI

RecJf

DNase I

Fluorescence Fluorescence

Fluorescence Fluorescence Electrochemical Electrochemical Electrochemical Electrochemical Chemiluminescence Chemiluminescence Chemiluminescence Chemiluminescence Colorimetry

TB IgE Membrane protein Adenosine TB TB Human IgG TB CCRF-CEM cells TB OTA CEA TB T-DNA2 Lysozyme

Fluorescence Fluorescence Fluorescence Fluorescence

Kþ OTA TB TB TB OTA TB

Electrochemical Electrochemiluminescence Electrochemical Electrochemical Electrochemical

Fluorescence Colorimetric Colorimetric Colorimetric Chemiluminescence Chemiluminescence

SERS

Electrochemical

Fluorescence

Xu et al. (2013) Liu et al. (2012a)

Hu et al. (2012) Lv et al. (2014) Liu et al. (2013a) Liu et al. (2014c) Yang et al. (2014b) Bao et al. (2015) Huang et al. (2012) Cai et al. (2013) Zhang et al. (2014d) Bi et al. (2014) Ma et al. (2014)

Tan et al. (2014) Li et al. (2012) Zhang et al. (2014e) Huang et al. (2013) Hun et al. (2013) Zong et al. (2014)

He et al. (2013)

50 nM 0.64 pg/mL 1.7 pM 20 fM 0.05 pM 0.12 pM 0.1 pM

Miao et al. (2014) Yang et al. (2014a) Yi et al. (2013) Peng et al. (2014a) Jiang et al. (2013c)

Lu et al. (2010a)

Wei et al. (2014) Tang et al. (2012a) Yan et al. (2013)

Bai et al. (2013a)

OTA Cocaine OTA Kanamycin OTA ATP IFN-λ

Fluorescence Electrochemical Electrochemical

40 nM 100 nM 0.2 nM 2.7 nM 20 nM 0.1 pM 0.065 pM

Exonuclease I

SEB TB

Fluorescence Electrochemical

0.3 pg mL  1 0.1 nM

Wu et al. (2013b) Jiang et al. (2013a)

T7 exonuclease

OTA Adenosine PDGF-BB

Fluorescence

25.2 fM 10.2 fM 75 aM

Huang et al. (2014)

S1 nuclease

L-argininamide

Chemiluminescence

100 nM

Hun and Wang (2012)

Nt.AlwI

Lysozyme Adenosine

Chemiluminescence SPR

0.2 pM 4 fM

Hun et al. (2010) Yao et al. (2014)

Fluorescence

region to form a duplex. As a result, Exo III hydrolytically digests the 3′-end strand, releasing the quencher, forming the fluorescent nucleic acid, and subsequently dissociating ATP. During this process, the ATP analyte is recycled, which then forms a complex with

Lin et al. (2014)

another nucleic acid hairpin in a free way. Therefore, the free fluorophore is autonomously formed in a cyclic manner, and an ATP molecule generates many FAM-modified fragments by fluorescence in this manner. As a result, the detection limit for ATP

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reached 0.25 μM. In the same study, Liu et al. applied the analyteinduced self-assembly of aptamer subunits and Exo III as an amplifying catalyst for the analysis of thrombin and cocaine (Fig. S1B; Supplemental information ). A label-free technology implies easy operation, low cost and non-interference to aptamer affinity. Xu et al. (2013) reported a new hairpin aptamer probe for label-free and sensitive fluorescent ATP detection based on Exo III-catalyzed target recycling signal amplification and SYBR Green I (SGI) indicator, as shown in Fig. S2 (Supplemental information). In the absence of ATP, the hairpin aptamer with five protruding mononucleotides at the 3′-terminus was resistant to Exo III digestion. The dsDNA-specific fluorescent SGI dye thus could be effectively intercalated into the stem of the hairpin structure and generate strong fluorescent emission at 520 nm. Upon the addition of ATP, it was bound to the corresponding aptamer sequence and the hairpin aptamer probe was reconstituted to form a new hairpin structure with a recessed 3′ terminus, which could be preferably cleaved by Exo III to release ATP and initiate Exo III-catalyzed target recycling signal amplification. This automatic cyclic process resulted in cleavage of a huge number of hairpin aptamers and greatly reduced intercalation of SGI into the stems of hairpin aptamer probes, causing obviously suppressed fluorescent signals for sensitive ATP detection. Moreover, the proposed method does not require a labeling or conjugation procedure and can be utilized in a homogenous solution. GO has been widely employed as a functional matrix for developing various fluorescent sensors, in which fluorophore-labeled ssDNA or aptamer could be adsorbed onto GO, leading to luminescence quenching of the fluorophores. Desorption of the probes from the GO through hybridization with complementary DNAs or binding with the aptamer target leads to fluorescence recovering (Liu et al., 2012a). Exo III was first introduced into the sensing system for amplified detection of DNA targets through the recycling of target DNAs (Liu et al., 2012a). Many non-nucleic acid detection methods have been converted into nucleic acid detection methods by using aptamers, and thus Exo III has been used for aptamer-based sensors for non-nucleic acid target detection. Hu et al. (2012) reported a GO-based fluorescent aptasensor for adenosine detection employing the Exo III cleavage function on the cDNA/signal dsDNA (Fig. S3; Supplemental information). Both the aptamer and the cDNA have four thymine nucleotides on their 3′ends to resist cleavage by Exo III (Brutlag and Kornberg, 1972). Without adenosine, adenosine aptamers were hybridized with cDNA, and Exo III could not split the single-stranded signal probes that were labeled with carboxyl fluorescein (FAM) at the 5′ end. Since final addition of GO, it could intensively adsorb singlestranded signal probes and effectively quenched fluorophores. On the contrary, the aptamers were associated with the targets if adenosine was present, leading to duplex DNA formation between cDNA and signal probes. Exo III could thereafter digest the duplex DNA from the 3′ blunt terminus of the signal probes, liberating the fluorophores. Upon GO addition, the adsorbing and quenching of fluorophores could not be achieved. With cyclic enzymatic cleavage coupled, a large fluorescent increase was witnessed and the LOD was obtained as low as 1 nM, lower than those of fluorescent aptamer sensors commonly used. There are also some other fancy fluorescent aptasensors based on Exo III-assisted signal amplification. However, they are complicated, not robust, expensive and unpractical, and therefore not discussed here (Lv et al., 2014). 2.1.2. Electrochemical methods Unlike the fluorescence-based optical detection method (Liu et al., 2013a), electrochemical methods can provide significant advantages, such as applications in simple and portable devices, rapid response and high sensitivity. Herein, some amplified

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biosensing systems were proposed with the electrochemical method and Exo III. Almost all electrochemical aptasensors involved a laborious and time-consuming step of immobilizing aptamers whose recognition to analytes is often restricted owing to the steric hindrance of the electrode surface compared with homogeneous assays. To develop homogeneous and highly sensitive electrochemical aptasensors, Liu et al. (2013a) developed a novel electrochemical aptamer-based strategy for ATP detection by combining Exo III-induced target recycling with the immobilizationfree electrochemical detection method (Fig. S4; Supplemental information). The proposed strategy took advantage of differential diffusivity between electroactive reporter tagged oligonucleotides and mononucleotides toward a negatively charged electrode surface. Thus, a hairpin-aptamer probe was ingeniously designed, which consisted of an aptamer sequence for ATP caged in the duplex structure of the stem and a ferrocene tag labeled at the 3′terminus. In the absence of ATP, the hairpin-aptamer probe can resist the cleavage activity of Exo III and exhibits a negligible electrochemical response on the sensing electrode. In the presence of ATP, the hairpin-aptamer probe binding to ATP results in the ferrocene tagged 3′-end to form a duplex. Then, Exo III hydrolytically digests the 3′-end strand, causing the release of ferrocenelabeled mononucleotides and subsequently the dissociation of ATP. Thus, a single ATP molecule enables several hairpin-aptamer probes to be digested and changed into more diffusive ferrocenetagged mononucleotides, thereby generating an amplified electrochemical signal. By the proposed method, a superior detection limit of 1 nM toward ATP with excellent selectivity can be achieved without any background subtraction. The “signal-on” high-performance electrochemical aptasensors offer a new direction in designing homogeneous, rapid, sensitive and selective detection of a wide spectrum of analytes. Based on the same ferrocene tag labeled hairpin probe as above, Liu et al. (2014c) developed another homogenous electrochemical biosensor for DNA and protein detection with an Exo IIIaided autocatalytic target recycling strategy in 2014. As shown in Fig. S5 (Supplemental information), Yang et al. (2014b) developed new highly sensitive label-free ECL detection of protein in combination with Exo III-assisted recycling amplification and a targetinduced DNA structure switching strategy (Fig. S6; Supplemental information). The dsDNA strands of S1/S2/thrombin aptamers at the sensing surface with 3′-blunt or 3′-protruding termini are resistant to Exo III digestion in the absence of thrombin, thus the ECL indicator, Ru(phen)32+ , can effectively intercalate into the grooves of the dsDNA strands and generate strong ECL emission. The association of the thrombin target with the corresponding aptamers releases the thrombin-aptamer from the sensor surface and switches the surface-immobilized dsDNA with 3′-protruding termini to those with 3′-recessing termini, leading to the formation of nicking sites for Exo III. The Exo III digestion releases the secondary target sequence (S2*, the cyan part of S2), which is complementary to the part of TBA. S2* hybridizes with thrombin aptamer to produce 3′-blunt termini to initiate Exo III-assisted cyclic digestion of the dsDNA strands by releasing more S2*, which significantly reduces the intercalation of Ru(phen)32 þ and generates inhibited ECL emission signals for thrombin detection. This Exo III-assisted amplified inhibition of ECL emission leads to highly sensitive detection of thrombin down to the level of 45 fM. There are also some other electrochemical aptasensors based on Exo III-catalyzed target recycling for signal amplification. As shown in Fig. S7 (Supplemental information), Bao et al. (2015) designed a hairpin DNA consisting of ATP aptamer sequences with MB labeled at the 5′-terminus. Upon the binding of ATP to aptamer, the MB-labeled hairpin DNA structure switched to the

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G-quadruplex and five protruding mononucleotides at the 5′-end hybridized with DNA immobilized on the gold electrode captured. As a result, MB became close to the electrode surface and showed a strong signal, and the 3′-end formed a double-stranded structure to create catalytic cleavage sites for Exo III. Then, the G-quadruplex structure was destroyed and ATP was released for target recycling. The proposed aptasensor showed a linear range of 0.1–20 nM with a LOD of 34 pM. 2.1.3. Chemiluminescence methods CRET relies on non-radiative dipole–dipole energy transfer from a chemiluminescent donor to a suitable acceptor molecule and promises an attractive optical detection scheme for bioanalytical applications (Huang et al., 2006; Zhang et al., 2009a). Huang et al. developed an Exo III-based CRET aptasensor for ultrasensitive detection of biomolecules. The principle of the amplified CRET aptasensor is outlined in Fig. S8 (Supplemental information) (Huang et al., 2012). The system is composed of an aptamer hairpin, an Exo III biocatalyst, and FAM labeled DNA-functionalized AuNPs, which act as signaling probes. The aptamer hairpin contains a 3′-terminus resistant to Exo III and is used to recognize and bind to the target. Luminol–H2O2–HRP and luminol–H2O2–HRP– FAM systems were used for CL production and CRET, respectively. In the latter system, luminol and FAM act as the donor and acceptor, respectively. If the target analyte is unavailable, AuNPs functionalized with FAM-labeled DNAs co-exist with the aptamer hairpin in the solution stably, and DNAs are not cleaved. After the solution is mixed with luminol, (an oxidizer) H2O2 and a catalyst HRP, CRET occurs between FAM and luminol, which produces CL. Due to the FAM–AuNP RET, AuNPs significantly quench the CRETstimulated emission of FAM. If the target analyte, aptamer hairpin and Exo III exist, however, the aptamer hairpin opens, binds to the target, and undergoes a change in its conformation. In this way, an aptamer–target complex is formed with a single-stranded sequence at its 5′-end, which hybridizes with FAM-labeled DNA related with AuNPs and develops into a DNA–DNA duplex in the resulting aptamer–target complex. Such a DNA–DNA duplex is formed to trigger enzymatic cleavage of FAM-labeled DNAs with Exo III selected, leading to FAM removal and aptamer–target complex release. Then, the released complex hybridizes with another FAM-labeled DNA, starting a new cycle, which causes further cleavage of FAM-labeled DNAs that are linked to AuNPs. Consequently, the aptamer–target complex can trigger FAM-labeled DNA cleavage multiple times, generating a parallel increase in the CRETstimulated FAM emission caused by the inhibition of FAM–AuNP interaction. This provides a path for amplifying CL detection of aptamer–substrate complexes. The estimated LOD of thrombin is 2 fM, much higher than that of many reported aptasensors. Cai et al. (2013) reported another exonuclease-assisted target recycling amplification strategy based on aptamers for sensitive CL determination of adenosine (Fig. S9; Supplemental information). The system contained amino-modified DNA sequences as capture probes, linker DNAs containing aptamer sequences as detection probes, and AuNP-functionalized DNA sequences (gold probes) as reporter probes. In the presence of adenosine, the linker DNA turned into a tertiary complex with a recessed 3′-terminus due to its structural switching property and was released from the capture probes immobilized on the surface of a 96-well plate, which minimized the possibility of hybridization with AuNP-functionalized reporters followed by a CL signal decrease. Exo III catalyzed stepwise mononucleotide removal from the 3-hydroxyl termini of duplex DNAs of aptamers and liberated the adenosine, which could then hybridize with a second aptamer on the 96-well plate and start another cycle. The experimental results revealed that the exonuclease-assisted recycling strategy provided a wide working range and a LOD (0.5 nM) for the monitoring of adenosine.

2.1.4. Colorimetric methods The GNP based aptasensor is one of the most popular colorimetric aptasensors since it possesses size- and distance-dependent optical properties. Ma et al. (2014) developed a GNP-based colorimetric aptasensor for detecting adenosine with Exo III-assisted recycling amplification (Fig. S10; Supplemental information). With this aptasensor, GNP probes functionalized with two kinds of single-stranded oligonucleotides were firstly hybridized with a complementary single-stranded oligonucleotide with the shape of adenosine aptamer, generating GNP aggregates (crosslinked GNPs in purple). With adenosine present, the linker formed dsDNA with a recessed 3′-end because of its structural switching property, leading to cross-linked GNP disassembly and a system color change from purple to red. However, upon Exo III addition, the dsDNA was digested with enzyme from the 3′-hydroxyl termini and the adenosine was liberated. The released adenosine could then interact with another linker and the cycle started a new. The sensitivity could be improved by 10 times compared with that without Exo III amplification. There are some other highly sensitive Exo III-based CL or colorimetric signal amplification methods for detection of various targets. However, the designs are specious with many cascade autocatalytic recycling amplification or reaction elements so that they are not practical in real analysis (Bi et al., 2014; Zhang et al., 2014d). 2.2. Nt.BbvCI Nt.BbvCI is a nicking endonuclease (Heiter et al., 2005; Morgan et al., 2000) that can recognize short specific DNA sequences and cleaves only one strand of DNA on a double-stranded DNA substrate. Recently, many sensitive nicking-enzyme-based signal amplification assays including fluorescence and electrochemical methods for target detection have been demonstrated. 2.2.1. Fluorescence methods Since the Nt.BbvCI substrate is a double-stranded DNA, aptamer-based non-nucleic acid target detection must be changed into nucleic acid detection. A common way is to design a hairpinstructured probe, which contains at least two parts: one is the aptamer for target bingding and the other is the reporter segment for hybridization with aptamer in the absense of a target. Upon the target addition, the hairpin undergoes a conformational switch and releases the reporter segment, which then hybridizes with a signal probe labeled with a fluorophore and a quencher attached at either terminal to form a double-stranded recognition sequence for Nt.BbvCI to digest. Then, the fluorescence signal dramatically increases due to complete separation of the fluorophore from the quencher. The released recognition aptamer–target complex with the reporter segment is then hybridized with another signal probe, and enzymatic recycling amplification is triggered. With this principle, Xue et al. (2012) have developed a sensitive and homogeneous aptasensor for thrombin detection (Fig. S11; Supplemental information). This method can specifically detect thrombin with a detection limit as low as 100 pM. To decrease the background signal, the signal probe was designed as a MB in the study of Feng et al. (2012) and a detection limit of 5 pM was obtained for IgE detection. With a similar method, Li et al. (2014) reported a highly sensitive homogeneous detection method for the membrane protein on single living cells by using the microfluidic system to produce the monodisperse droplet as an independent microreactor for aptamer and nicking-enzyme-assisted fluorescence signal amplification. Huang et al. (2015b) developed an amplified FP aptasensor based on NESA and GO enhancement. As shown in Fig. S12 (Supplemental information), the aptamer contains a 10-nt extension

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sequence at its 5′-end and a fluorophore-labeled DNA probe partly complementary to the short extension sequence of the aptamer, it also contains the cleavage site for Nb.BbvCI. The aptamer is adsorbed onto the GO surface. Thus, the FAM dye exhibits a very high FP value due to the extraordinarily large volume of GO. Upon the target addition, the formation of the aptamer–target complex induces stable hybridization of an aptamer with a fluorophore-labeled DNA probe linked to GO, which forms a nicking site-containing duplex DNA region due to the enhancement of base stacking. Then, the subsequent Nb.BbvCI digestion results in the release of a short DNA fragment carrying the FAM dye from the GO surface and the intact target/aptamer complex can then hybridize with another fluorophore-labeled DNA probe. The continuous release of many short DNA fragments carrying the FAM dye from the GO generates a substantial decrease in the FP value, and thus achieving the target-assisted dual signal amplfication. Tan et al. (2014) have developed a sensitive proximity extension and the enzyme-assisted fluorescence method for protein detection. However, the method requires a pair of aptamers for one protein target and involves laborious procedures like nicking and polymerization, and therefore it is impractical and not worthy of promotion. 2.2.2. Colorimetric methods Using a colorimetric sensor, the target concentration can be determined by color change. This method is greatly desirable for in-field and on-site detection because expensive analytical instruments may become unnecessary. Similar to the aforementioned fluorescence method, it is common for a Nt.BbvCI-based signal amplification aptasensor to design a hairpin-structured probe. As shown in Fig. S13 (supplemental information), the signal probe was designed as a linker DNA which can assemble into two sets of DNA-modified AuNPs, inducing the aggregation of AuNPs (Li et al., 2012). In the presence of the target, the linker DNA was digested and could no longer assemble into the DNA-modified AuNPs. Thus, a red color of separated AuNPs could be observed. This method can be used to detect human thrombin and ATP with a detection limit of 50 pM and 100 nM by the naked eye. Its sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. Based on the same principle, Zhang et al. (2014e) developed a visual and sensitive detection method for cancer cells. The method could detect as few as 40 cells with a concentration ranging from 102 to 104 cells. Huang et al. (2013) developed a label-free colorimetric aptasensor based on nicking-enzyme-assisted signal amplification by designing a signal probe as a G-riched DNA probe containing two G-riched DNAzyme segments (Fig. S14; Supplemental information). The hybridization of the G-riched DNA with the blocker DNA prohibits the formation of the activated DNAzymes in the absence of targets. After Nt.BbvCI digestion, the G-riched DNAzyme segments interact with hemin and generate activated DNAzymes that can catalyze the H2O2-mediated oxidation of 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS2  ) to the colored ABTS  , thus enabling amplified colorimetric detection of targets. Using TB as a proof-of-principle analyte, this sensing platform can detect TB specifically with the LOD as low as 1.5 pM, which is at least 4 orders of magnitude lower than the unamplified colorimetric assay.

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oligonucleotide hybridization, which presents a high demand for the DNA design and reaction system. It is less practicable and will not be detailed here. 2.3. RecJf exonuclease RecJf is a recombinant fusion protein of RecJf and MBP, and it has the same enzymatic properties as wild-type RecJf while MBP enhances the RecJf solubility. It is a ssDNA-specific exonuclease (Jiang et al., 2013b), which can catalyze the removal of deoxynucleotide monophosphates from ssDNA in the 5′-3′ direction without requiring a 5′ phosphate. When the DNA containing a 22base 5′-extension is used as a substrate for RecJf, the resulting products are a mixture of DNA fragments that have blunt-ends, 5′ extensions (1–5 nucleotides) and recessed 5′ ends (1–8 nucleotides). 2.3.1. Electrochemical method Miao et al. (2014) developed an electrochemical aptasensor for potassium detection by using RecJf exonuclease mediated signal amplification (Fig. S15; Supplemental information). A DNA probe with a stem–loop structure containing an anti-K þ aptamer sequence is designed and immobilized on a gold electrode through a 3′-end thiol group. The 5′-end MB provides significant electrochemical signals. K þ can bind to the aptamer specifically to form a G-quadruplex structure, which splits the original stem–loop structure. RecJf exonuclease can then digest the induced singlestranded 5′-end, releasing the K þ aptamer, which can be combined to another DNA probe at the electrode surface. After RecJf exonuclease cleavage is triggered by K þ for many cycles, the intensity of electrochemical signals is sharply declined and can be utilized to determine the K þ concentration. Ruo′s group has combined RecJf-catalyzed target recycling and HCR or DNA concatemers for signal amplification in electrochemical assay for TB and OTA (Bai et al., 2013a; Jiang et al., 2013c; Peng et al., 2014a; Yi et al., 2013). However, these methods involve multiple sophisticated DNA probe designs and laborious preparation procedures, and therefore are not detailed in this review. 2.3.2. Electrochemiluminescence method Yang et al. (2014a) developed a highly sensitive electrochemiluminescence (ECL) aptasensor for OTA detection by using RecJf-catalyzed target recycling amplification. As shown in Fig. S16 (Supplemental information), duplex DNA probes containing biotin-modified aptamers are immobilized on a CdTe QD electrode coated with a composite film. The presence of the OTA target leads to effective removal of biotin-aptamers from the electrode surface, and the aptamers are then digested by the RecJf exonuclease to release the OTA target to initiate an OTA recycling process. The removal of a large number of biotin-modified aptamers prevents the attachment of STV–ALP through biotin–STV interaction. The ET from the CdTe QD in the excited state to the electro-oxidized species of the ALP enzymatic products during a potential scan is thus blocked. The QD ECL emission is recovered for quantitative OTA detection. The target recycling amplification was catalyzed by the exonuclease, so the ET blocking effect is significantly enhanced, realizing sensitive detection of OTA at the level of as low as 0.64 pg mL  1. 2.4. DNase I

2.2.3. Other methods Hun et al. (2013) and Zong et al. (2014) have developed nicking-endonuclease-based signal amplification for OTA or protein CL detection. He et al. (2013) have developed an ultrasensitive SERS detection of lysozymes using a nicking-endonuclease-based signal amplification aptasensor. However, these methods involve multi-

DNase I is a deoxyribonuclease that can digest ssDNA and dsDNA (Rosner, 2011; Yasuda et al., 2010). 2.4.1. Fluorescence methods Lu et al. (2010a) developed an amplified aptamer-based assay

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using graphene and DNase I (Fig. S17; Supplemental information). Graphene sheets can strongly bind to ssDNA through π-stacking interactions between nucleobases and graphene, which protect aptamers from nuclease cleavage. Furthermore, graphene sheets can quench the fluorescence of fluorophores conjugated with aptamers due to excellent electronic conductivity of graphene. After binding to the target, aptamer folds into a rigid structure and is dissociated from the graphene, whereupon the nuclease can split the free aptamer, thereby liberating the fluorophore and ultimately releasing the target. The released target then binds to another aptamer to trigger recycling signal amplification. Using this principle, the LOD for ATP was optimized to 40 nM from 10 mM when the 1:1 binding strategy was used without amplification. A similar DNase I-based amplification assay for ATP was developed by Lin et al. (2014) using carbon nanoparticles instead of graphene and the LOD was increased to 0.2 nM from 10 mM without amplification. Recently, nano-graphite has attracted considerable attention due to its ultra-high surface area for binding aptamers, low cost and production convenience in comparison with GO. They have similar mechanisms for quenching fluorophores and good biocompatibility and low cytotoxicity. With the same DNase I-based amplification principle, Wei et al. (2014) have detected OTA with a LOD of 20 nM using nano-graphite instead of graphene or carbon nanoparticles.

A novel and simple FRET bioassay was developed by Wu et al. (2013b. They ingeniously utilized two DNA fragments labeled with both fluorescence donor probes and quencher probes which were hybridized with a SEB aptamer to quench the fluorescence of UCNPs via FRET. In the presence of the target, the two DNA fragments separated from the aptamer because of hybridization between the target and the aptamer subsequently restored the UCNPs′ fluorescence. In addition, the SEB was liberated from the aptamer–SEB complex using exonuclease I for analyte recycling by selectively digesting particular DNA (SEB aptamer). They detected the SEB with a low LOD of 0.3 pg mL  1. This proposal was shown in Fig. S20 (Supplemental information). Jiang et al. (2013a) proposed an Exo I-assisted background noise reduction signal amplification strategy for thrombin detection by DET of hemin (Fig. S21; Supplemental information). The TB-binding aptamers (TBAs) as well as 6-mercapto-1-hexanol (MCH) are self-assembled on a sensing electrode surface through Au–S interaction. Without the target thrombin, the randomly coiled strands of TBA are digested by Exo I, preventing the association with hemin and significantly reducing the existing background noise. When thrombin is present, the folded TBA G-quadruplex is stabilized, which prevents it from degrading by Exo I. Therefore, the G-quadruplex bound to hemin generates amplified output signals.

2.4.2. Electrochemical methods Besides fluorescence methods, carbon nanostructure-based aptamer protection from nuclease digestion is also used to develop electrochemical methods for detection of various targets. Tang et al. (2012a) reported a label-free impedimetric detection of ATP by using DNase I cleavage induced target recycling on a sensing platform based on CNTs (Fig. S18; Supplemental information). This process involves immobilization of carbon nanotubes and aptamers on a glassy carbon electrode (GCE). The addition of targets dissociates the aptamer from CNTs and the addition of DNase I triggers digestion of the free aptamer and release of the target to start new recycling. Similarly, little target ATP can induce release of many aptamers efficiently from the aptasensors, which leads to a substantial decrease in EIS-monitored electron transfer resistance (Ret). Yan et al. (2013) reported another label-free electrochemical aptasensor for detection of IFN-λ based on a graphene-controlled assembly and DNase I-assisted target recycling signal amplification strategy. As shown in Fig. S19 (Supplemental information), in the absence of IFN-λ, graphene could not be assembled onto the 16MHA-modified gold electrode because the IFN-λ binding aptamer was strongly adsorbed on the graphene due to the strong π–π interaction. Thus, the electronic transmission was blocked (eT OFF). However, the presence of target IFN-λ induces desorption of aptamers and further DNase I digestion of aptamer release targets for another aptamer binding, resulting in successive release of aptamers from the graphene. Then, the “naked” graphene could be assembled onto the MHA-modified gold electrode with π-conjunction, mediating the ET between the electrode and the electroactive indicator. The developed label-free electrochemical aptasensing technology showed a linear response to the concentration of IFN-λ ranging from 0.1 to 0.7 pM with the LOD of 0.065 pM.

2.5.2. T7 exonuclease T7 Exo can catalyze removal of mononucleotides from the 5′hydroxy termini of duplex DNAs step by step, thereby enabling selective digestion of one strand in the double-stranded DNA structure. This useful property has been investigated to develop T7 Exo-based sensing platforms for signal amplification (Kerr and Sadowski, 1972; Wang et al., 2014). Huang et al. (2014) reported an alternative, an attractive ultrasensitive fluorescence polarization aptasensor relying on T7 Exo signal amplification and PS NP amplification. A DNA probe modified with FAM at its 5′-end was linked to PS NPs at the 3′-end and had a very high FP value due to the much larger volume of PS NPs. With an appropriate design of aptamer/DNA pairs or aptamer subunits, the aptamer could hybridize with FAM-labeled DNA probes and trigger T7 Exo digestion of FAM-labeled probes upon target binding, resulting in removal of the FAM dye from PS NPs and a decrease in the FP value. The aptamer–target complex was also released to hybridize with the next DNA probe initiating a signal amplification cycle. As shown in Fig. S22 (Supplemental information), ultra-high sensitivity for OTA detection was obtained with a detection limit of 250 aM. With the same amplification based on T7 Exo-PS NP but a different design of aptamer/DNA pairs or aptamer subunits, the authors (Huang et al., 2014) also developed two other alternative strategies for ultrasensitive detection of ATP and platelet-derived growth factor (PDGF).

2.5. Other nucleases 2.5.1. Exonuclease I (Exo I) Exonuclease I breaks ssDNA in the 3′-5′ direction (Goellner et al., 2015), releasing deoxyribonucleoside 5′-monophosphates one after another. It does not cleave DNA strands without terminal 3′-OH groups because they are blocked by phosphoryl or acetyl groups.

2.5.3. S1 nuclease S1 nuclease (Shimada et al., 2015; Vogt, 1973) is an endonuclease that is active against single-stranded DNA and RNA molecules. It can hydrolyze single-stranded regions such as loops and gaps in duplex DNA. More importantly, a specific recognition site and hydrolysis in the 3′–5′ (or 5′–3′) direction are not needed for the S1 nuclease, and therefore hydrolysis occurs despite the sequence and direction at the terminus or loops. This is in sharp contrast with the nicking endonucleases employed in the above amplification schemes. Using S1 nuclease hydrolysis on the single-stranded regions of an aptamer–target complex, Hun and Wang (2012) introduced a novel scheme of signal amplification to increase the detection sensitivity of L-argininamide. As shown in Fig. S23 (Supplemental information), DNA1 (complementary sequence of L-argininamide

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aptamer) labeled with the 5′-CL reagent N-(4-aminobutyl)-Nethyl-isoluminol (ABEI) is immobilized on MBs coated with colloid gold. 3′-ABEI-labeled DNA2 (L-argininamide aptamer) is self-assembled on MBs in duplexes. With L-argininamide, the DNA1 aptamer is liberated from complementary DNA2 and takes a structural switch from the duplex to the stem–loop aptamer structure, and the single-stranded regions are formed in DNA2. Thus, the S1 nuclease catalyzes the stepwise removal of mononucleotides from the single-stranded regions of the aptamer as well as the forming of the single-stranded DNA1 on the MB surface, and then the released L-argininamide interacts with another aptamer, whence the cycle starts a new. An L-argininamide could generate many CL reagents ABEI in this way, and a large amount of released ABEI reagents could be sensitively determined in the ABEI–DPN–isoniazid reaction system, generating strong CL signals. 2.5.4. Nt.AlwI Nt.AlwI is a nicking endonuclease that cleaves only one strand of a DNA on a dsDNA substrate (Chen and Zhao, 2013; Zhuang et al., 2014). Even though Nt.AlwI has also been used to develop signal amplification technologies for chemiluminescence or surface plasma resonance aptasensors (Hun et al., 2010; Yao et al., 2014), the approaches involve multiplex single DNA hybridization procedures and are therefore impractical.

3. Summary and conclusions We have summarized recent advances in the development of target recycling signal amplification aptasensors that employ nuclease digestion on the aptamer. The key in these assays is to design proper aptamer probe which can be digested after binding to the target and then release the target to initiate an signal recycling process. To address this, various fluorescence, colorimetry, electrochemistry and chemiluminescence based strategay have been developed by using different nuclease with unique digestion properties. Compared with polymerase-based signal amplification in aptasensors, nuclease-based signal amplification aptasensors are simpler without complicated DNA probes or primer design and multi-step reaction, avoiding possible false positive amplification. There are multiple types of nucleases which show unique cleavage properties with different recognition sites and substrates. For examle, NEases (Kiesling et al., 2007; Morgan et al., 2000; Zheleznaya et al., 2009) (Nt.BbvCI and Nt.AlwI) recognize specific nucleotide sequences in dsDNA and catalyze the cleavage of only one strand at a fixed position. The high specificity has resulted in an increased detection limit compared with other nucleases apart from restricting target detection varieties. It is worth mentioning that the sensitivity of aptasensors of the colorimetric method based on Nt.BbvCI has achieved the expected goal by naked eyes, which provides a new approach for rapid detection. Unlike nicking endonucleases, Exo III is sequence-independent and does not require a specific recognition site, providing a great potential for highly sensitive, selective and simple detection of a wide range of target molecules. DNase I is a deoxyribonuclease that can digest ssDNA and dsDNA and is appropriate for macromolecules, which have been directly recognized by aptamers without the hairpin structure or other conditions. Other nucleases are not widely used, and specific features have been set out in Table 1. Occasionally, two or three nucleases (He et al., 2013; Ma et al., 2012) are simultaneously used in one aptasensor, however, these methods are tedious, complex and not applicable so that they are not detailed in this review.

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4. Future perspectives To develop highly sensitive and specific bioanalytical techniques to achieve simplicity and high throughput is critically demanded for medical diagnosis, environmental monitoring and food safety analysis. Aptamer, with its unique nucleic acid properties, is a powerful tool to develop highly sensitive and homogeneous analysis approaches compared to conventional antibodybased immunoassay, which usually involves multi-step washing and lacks signal amplification techniques. Taking the advantages of nucleases, aptamer-based sensing reactions occur in isothermal and homogeneous conditions that facilitate both target recognition and signal amplification in the solution. In addition, the target can be easily detected using common detection modalities, which include electrochemistry (Cao et al., 2012; Liu et al., 2013b), fluorescence (Lu et al., 2010b; Xue et al., 2012), chemiluminescence (Bi et al., 2014), and colorimetry (Li et al., 2012). Moreover, the applications of aptamers to assays can overcome the limitation of many homogeneous binding assays that focus on DNA detection, by broadening the scope of detection of proteins, small molecules and ions. However, the development and routine applications of nuclease-based aptasensor-mediated homogeneous binding assays are confronted with several challenges. Newly developed assays are approved in clinical laboratories and commercialized in a slow pace. (i) Most of the assays were developed and tested in wellcontrolled buffer systems, not in biological fluids or cells. Because there are no separation steps in homogeneous binding assays, matrix effects of the samples, such as those from human serum or plasma, need to be evaluated carefully. Further optimization or modification should be made when nucleic acids and enzymes are used in routine analysis because they may become unstable in the presence of a particular sample matrix. (ii) Similar signal generation and amplification strategies have been adopted to only a few targets with well-understood aptamer structures, including human thrombin, ATP, cocaine, adenosine. Therefore, it is seriously required to develop universal molecular translators that can convert the information about other practical targets into an amplifiable and detectable output signals. (iii) It is difficult to adapt current nuclease-based signal amplification assays to the determination of multiple targets simultaneously. Simultaneous detection of multiple targets using homogeneous binding assays requires minimum or no cross-reaction between the multiple affinity aptamers and the various targets when the multiple affinity ligands are present in the same test solution. (iv) Even many different nuclease-based signal amplification strategies have been developed for highly sensitive detection of various targets, however, some of these technologies are harder to generalize as they are over-elaborate designed such as fine-tuning probes. Finding easier, more effective solutions are still urgently needed to meet the growing demand for the sensitivity in biomedical, environmental and food analysis. Confronting these challenges presents new opportunities for further research and development on nuclease-based signal amplification aptasensors, in particular better selection of stable aptamers and innovative design of nuclease-based target recycling approach. While our aim is to provide a general guidance for the design of nuclease-based signal amplification biosensing assays, we also hope that the strategies of nuclease-mediated target recycling discussed herein can be applied in other aptamer-based sensing platforms, such as surface plasmon resonance (SPR), resonance scattering and some DNAzyme based approaches in which an aptamer-like DNAzyme probe is used for special target binding. We also hope that these principles can facilitate research in nucleic acid mediated homogeneous assays, where nucleic acids are used as either the recognition component for nucleic acid

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targets such as molecular beacons and nanomaterial-based assays. In addition to these nuclease reviewed here, we also hope other nucleic acid-cleaving enzymes, including restriction endonucleases (Weizmann et al., 2006), RNase H (Harvey et al., 2004) , and apurinic–apyrimidinic-based endonuclease IV (Xiao et al., 2012) which have been used for nucleic acid target detection, could also be applied in aptamer-based homogeneous assay for recycling targets and generating cyclic amplification. Also, as these proof-of concept experiments show, the nuclease-based signal amplification aptasensors could enable the detection of various molecules for which an aptamer is identified by SELEX. Due to its simple design, easy operation, fast response and high sensitivity and selectivity, the proposed strategy could be expected to find more applications in clinical diagnostics, food safety test and environmental pollutant analysis.

Acknowledgments This work was supported by International Science & Technology Cooperation Program of China (2012DFA31140). The authors express their gratitude for the support.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.10.015.

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