Label-Free and Enzyme-Free Homogeneous Electrochemical

Nov 2, 2015 - Homogenous electrochemical biosensing strategies have attracted substantial attention, because of their advantages of being immobilizati...
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Label-Free and Enzyme-Free Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive and Highly Specific MicroRNA Assay Ting Hou, Wei Li, Xiaojuan Liu, and Feng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02790 • Publication Date (Web): 02 Nov 2015 Downloaded from http://pubs.acs.org on November 2, 2015

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Analytical Chemistry

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Label-Free and Enzyme-Free Homogeneous Electrochemical

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Biosensing Strategy Based on Hybridization Chain Reaction: A Facile,

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Sensitive and Highly Specific MicroRNA Assay

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Ting Hou,§ Wei Li,§ Xiaojuan Liu, and Feng Li*

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College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University,

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Qingdao 266109, People’s Republic of China

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§

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* Corresponding author. Tel/Fax: 86-532-86080855

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E-mail: [email protected]

T. Hou and W. Li contributed equally to this work.

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ABSTRACT

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Homogenous electrochemical biosensing strategies have drawn substantial attention due to their

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advantages of immobilization free, rapid response and improved recognition efficiency as compared to

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heterogeneous biosensors, but the high cost of labeling and the strict reaction conditions of tool enzymes

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associated with current homogeneous electrochemical methods limit their potential applications. To address

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these issues, herein we reported, for the first time, a simple label-free and enzyme-free homogenous

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electrochemical strategy based on hybridization chain reaction (HCR) for sensitive and highly specific

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detection of microRNA (miRNA). The target miRNA triggers the HCR of two species of metastable DNA

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hairpin probes, resulting in the formation of multiple G-quadruplex-incorporated long duplex DNA chains.

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Thus, with the electrochemical indicator methylene blue (MB) selectively intercalated into the duplex DNA

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chain and the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is

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depended on the concentration of the target miRNA. Thus, by this “signal-off” mode, a simple, label-free

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and enzyme-free homogeneous electrochemical strategy for sensitive miRNA assay is readily realized. This

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strategy also exhibits excellent selectivity to distinguish even single-base mismatched miRNA.

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Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both

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expensive labeling and sophisticated probe immobilization processes are avoided. Therefore, the

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as-proposed label-free and enzyme-free homogeneous electrochemical strategy may become an alternative

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method for simple, sensitive and selective miRNA detection, and has great potential to be applied in

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miRNA-related clinical diagnostics and biochemical researches.

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Analytical Chemistry

INTRODUCTION

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MicroRNAs (miRNAs), small single-stranded noncoding RNAs (containing approximately 19–23

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nucleotides), can influence many biological processes by regulating gene expression via mediating

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translational repression or promoting degradation of target messenger RNAs (mRNAs).1,2 To date, over

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1000 human miRNAs have been identified, which can target more than 30% of the human genome.3 Apart

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from being post-transcriptional regulators, accumulating evidences have proved that abnormal expression

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of certain miRNAs is closely related to a variety of diseases and disorders, especially cancers. Thus

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miRNAs have been regarded as potential targets in disease diagnosis and therapy, as well as new

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biomarkers for diseases such as cancers, cardiovascular and autoimmune diseases.3–6 However, miRNA

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detection is challenged by the characteristics of miRNA, including small size, sequence homology among

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family members, low abundance in total RNA samples, and susceptibility to degradation.3,7 Therefore,

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strategies for sensitive and selective detection of miRNAs are in urgent need, especially for biomedical

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research and early clinical diagnosis.

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Several traditional methods have been exploited to detect miRNAs, including Northern blotting,8

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real-time PCR,9 and microarrays.10 However, these methods have some intrinsic limitations, such as low

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sensitivity, poor specificity and long assay time. To overcome the shortcomings of the traditional methods,

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great efforts have been made to develop colorimetric,11 fluorescent12–14 and electrochemical methods15–17

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for miRNA detection. Among them, electrochemical miRNA assays have attracted much attention due to

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the advantages of low cost, simple operation, miniaturizability and fast response. For example, Ai’s group

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reported an one-step, ultrasensitive electrochemical miRNA assay based on T7 exonuclease-assisted cyclic

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enzymatic amplification.15 Sun’s group realized ultrasensitive electrochemical detection of miRNA based

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on an arched probe-mediated isothermal exponential amplification.16 However, the aforementioned 3

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electrochemical methods all needed the immobilization of DNA probes on the electrode surface, which

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suffered from some intrinsic drawbacks: (1) the immobilization processes are complicated and

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time-consuming, (2) the immobilization procedures may affect the activity of the immobilized

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biomolecules, and (3) the limited reaction area and the existence of local steric hindrance may lead to

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relatively low binding efficiency.18,19 Thus, it is highly desirable to develop faster and simpler

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immobilization-free electrochemical methods to assay miRNAs. Recently, a variety of homogeneous

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electrochemical strategies have been developed for the detection of various targets, including DNA, small

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biological molecules, metal ions and enzyme activities.18–28 For example, Xuan et al. demonstrated a

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solution-phase electrochemical strategy for sensitive detection of DNA and mercury ion.20,21 Our group

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developed homogeneous electrochemical strategies for highly sensitive detection of ATP, DNA

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methyltransferase activity, alkaline phosphatase activity, and telomerase activity.19,22,25,27,28 These

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homogenous electrochemical methods have successfully realized rapid, highly sensitive and selective

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detection of various targets, but the relatively high cost of the labeling of the oligonucleotides and the strict

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reaction condition of the tool enzymes limit their wider applications. Thus, it is highly desirable to develop

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label-free and enzyme-free homogeneous electrochemical strategies. Methylene blue (MB), is a

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positively-charged organic dye with aromatic structure, and can be used as an electrochemical indicator.20,29

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Previous studies have demonstrated that MB can intercalate into double-stranded DNA (dsDNA) through

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π-π stacking interactions.30 Very recently, Zhang et al. found that the binding between MB and

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G-quadruplex was even stronger than that between MB and dsDNA, and a label-free homogenous

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electrochemical biosensor based on the formation of G-quadruplex/MB complex has been developed.23

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Thus, by taking advantage of its unique ability to bind with both G-quadruplex and dsDNA, MB can be

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readily adopted in label-free homogenous electrochemical assays. 4

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Nowadays, many signal amplification strategies have been developed for sensitive detection of

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miRNAs, including hybridization chain reaction (HCR) amplification,13,17 rolling circle amplification

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(RCA),12 and isothermal exponential amplification.14,16 For instance, Li’s group reported a graphene

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surface-anchored fluorescence sensor for sensitive detection of miRNA coupled with enzyme-free signal

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amplification of HCR.13 Tang’s group reported highly specific and ultrasensitive isothermal detection of

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miRNA by padlock probe-based exponential rolling circle amplification.12 Ye’s group reported

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ultrasensitive miRNA detection by DNA-scaffolded silver-nanocluster probe based on isothermal

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exponential amplification.14 Among them, HCR amplification shows great potential in signal amplification

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due to its unique features: (1) HCR is a kinetics-controlled reaction in which a cascade of hybridization

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events between two species of metastable DNA hairpin probes are triggered to form a long dsDNA

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structure, and it does not require any enzymes; (2) the HCR amplification shows high sensitivity and

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selectivity toward target detection; (3) the process of HCR is simple and can be achieved under mild

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experimental conditions.13,31–37 It is also worth noting that, very recently Hsing and coworkers reported a

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novel enzyme-free and hairpin-free fluorescence sensing strategy based on nonlinear HCR amplification,38

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and further utilized this strategy to realize highly sensitive immobilization-free electrochemical detection of

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nucleic acids.39 In consideration of its advantages, we believe that HCR amplification strategy is an ideal

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candidate for signal amplification in label-free homogeneous electrochemical sensing.

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Inspired by the aforementioned works, herein, we reported for the first time a label-free and

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enzyme-free homogeneous electrochemical biosensing strategy for sensitive detection of miRNA based on

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HCR signal amplification. In this strategy, two DNA hairpin probes (HP1 and HP2) were ingeniously

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designed. HP1 contained sequences partially complementary to target miRNA, as well as to HP2. Moreover,

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split G-quadruplex (3:1) sequences were present at both ends of HP1 and HP2. In the absence of target 5

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miRNA, HP1 and HP2 co-existed in solution, with only small portion of MB intercalated into their stems,

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thus large amount of free MB in solution exhibited large diffusivity towards the indium tin oxide (ITO)

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electrode surface, resulting in big electrochemical signal. However, in the presence of target miRNA, HP1

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was unfolded via the hybridization with miRNA to trigger the autonomous cross-opening of the two hairpin

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probes through the toehold-mediated strand displacement reaction, and an HCR product was formed, with

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multiple G-quadruplexes incorporated on both sides of the long duplex DNA chain. As a result, with MB

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selectively intercalated into the dsDNA and G-quadruplex, the diffusion current of MB was largely reduced,

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and the repulsion between the negatively charged ITO electrode surface and the long duplex DNA chain

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further prevented the intercalated MB from reaching the electrode surface, thus a significant

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electrochemical signal drop was observed, which is proportional to the concentration of the target miRNA.

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By this “signal-off” mode, label-free and enzyme-free homogeneous electrochemical strategy based on

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HCR amplification for sensitive miRNA assay was readily realized.

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EXPERIMENTAL SECTION

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Reagents. HPLC-purified miRNA and diethylprocarbonate (DEPC)-treated deionized water were

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obtained from GenePharma Co., Ltd. (Shanghai, China). HPLC-purified DNA oligonucleotides and

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ribonuclease (RNase) inhibitor were purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai,

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China). Tris(hydroxymethyl)aminomethane (Tris), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

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(HEPES), methylene blue (MB),hydrochloric acid (HCl), NaCl, KCl and MgCl2 were purchased from

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Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), which were of analytical grade and used

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without further purification or treatment. Before use, miRNAs were diluted to appropriate concentrations

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with DEPC-treated water. The DNA hairpin probes were diluted with 10 mM HEPES (containing 100 mM

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NaCl, 25 mM KCl, 10 mM MgCl2, pH 7.0), and the ssDNAs were diluted with 10 mM Tirs-HCl 6

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(containing 50 mM NaCl, 10 mM MgCl2, pH 7.4), to give the stock solutions. DEPC-treated deionized

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water was used in all experiments. The sequences of the oligonucleotiedes were listed in Table 1.

Table 1. Sequences of the oligonucleotides used in the experimentsa

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Name

Sequence (from 5′ to 3′)

let-7a

5′-UGA GGU AGU AGG UUG UAU AGU U-3′

let-7f

5′-UGA GGU AGU AGA UUG UAU AGU U-3′

let-7g

5′-UGA GGU AGU AGU UUG UAC AGU U-3′

HP1

HP2

5′-AGG GCG GGT GGG TTG TAT AGT AGG CAA AGT AAC TAT ACA ACC TAC TAC CTC ATG GGT-3′ 5′-TGG GTA CTT TGC CTA CTA TAC AA T GAG GTA GTA GGT TGT ATA GTA GGG TAG GGC GGG-3′

DNA1

5′-GGG TTG GGC GGG ATG GGT-3′

DNA2

5′-ACC CAT CCC GCC CAA CCC-3′

(complementary to DNA1)

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a

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and HP2, the boldface letters represent the sequences complementary to each other to form the stems of the

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hairpin probes, respectively. The underlined letters in HP1 and HP2 represent the two segments of the

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complementary sequences between the two hairpin probes.

In HP1, the letters in italics represent the sequences complementary to the target miRNA (let-7a). In HP1

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Electrode pretreatment and electrochemical measurement. The indium tin oxide (ITO)

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electrode was pretreated by being sequentially sonicated in an Alconox solution (8 g of Alconox per liter of

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water), propan-2-ol, acetone, and ultrapure water for 15 min each. Then, the ITO electrode was immersed

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in 1 mM NaOH solution for 5 h at room temperature and sonicated in ultrapure water for 15 min. After

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these cleaning procedures, a negatively charged working electrode surface was obtained and was ready to

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use. Differential pulse voltammetric (DPV) measurements were performed using a CHI 660E

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electrochemical analyzer (Shanghai, China), with the potential sweeping from −0.6 to 0.1 V, and the pulse 7

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height, the pulse width, the pulse increment and the pulse period set to 0.05 V, 0.05 s, 0.004 V and 0.5 s,

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respectively. A three-electrode system was employed, with an ITO electrode with an active surface area of

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0.12 cm2 as the working electrode, an Ag/AgCl as the reference electrode, and a platinum wire as the

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auxiliary electrode.

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HCR-Amplified Homogeneous Electrochemical miRNA Assay. HP1 and HP2 were

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pretreated by being heated to 95 °C and incubated at this temperature for 5 min, and slowly cooled down to

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room temperature. The miRNA assay was performed in 50 µL of miRNA reaction buffer (10 mM HEPES,

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100 mM NaCl, 25 mM KCl, 10 mM MgCl2, pH 7.0) containing 0.5 µM HP1, 0.5 µM HP2, 10 µM MB, 10

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U of RNase inhibitor, 1 mM DTT and the target miRNA with different concentrations, and the reaction

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solution was incubated at 37 °C for 2 h before the electrochemical measurements. The control experiment

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was carried out under the same condition without adding miRNA. All experiments were repeated three

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times. To minimize the effect of RNase on the stability of miRNAs, all glassware, pipette tips and

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centrifuge tubes used in the experiments were autoclaved using Tomy SX-500 Autoclave (Tokyo, Japan).

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RESULTS AND DISCUSSION

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Principle of Homogeneous Electrochemical miRNA Assay. The principle of the

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HCR-amplified homogeneous electrochemical strategy for sensitive detection of miRNA is illustrated in

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Scheme 1. Two DNA hairpin probes, namely HP1 and HP2, are ingeniously designed. In HP1, the

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sequences complementary to the target miRNA (let-7a) are partially embedded in its stem, which are also

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partially complementary to the sequences embedded in the stem and loop of HP2. HP1 and HP2 have

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additional segments complementary to each other. Moreover, split G-quadruplex (3:1) sequences are

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present at both ends of HP1 and HP2. In the absence of the target miRNA, both HP1 and HP2 maintain

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their hairpin structures in the solution. With methylene blue (MB) being added, small portion of MB 8

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intercalates into the stems of HP1 and HP2, while the majority of MB is free in solution, which shows high

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diffusivity towards the ITO electrode surface, thus resulting in a relatively large electrochemical signal.

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However, when the target miRNA is present, the following HCR process is triggered. First, the target

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miRNA unfolds HP1 via the toehold-mediated strand displacement reaction. Next, the newly exposed

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sticky sequences (in purple) in HP1 hybridize with the purple segment in HP2 to unfold HP2. Then, the

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newly released sticky sequences (in green) in HP2, in turn, hybridize with the green segment in another

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intact HP1. In this manner, miRNA initiates the chain-like assembly of HP1 and HP2 through HCR,

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generating a long chain of duplex DNA. Moreover, upon the formation of the long duplex chain, multiple

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HP1s are connected in a head-to-tail fashion in one chain, and HP2s in the other chain. In this way, the split

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G-quadruplex sequences from adjacent HP1s (as well as HP2s) are brought close to each other, and with

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the aid of potassium ions, multiple G-quadruplexes are formed on both sides of the duplex chain. Upon the

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intercalation of MB into the long duplex DNA chain as well as the multiple G-quadruplexes, the MB

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molecules are “locked” into the long multiple G-quadruplex-incorporated duplex DNA chain, whose

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diffusivity is significantly reduced.23 Moreover, due to the electrostatic repulsion between the negatively

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charged ITO electrode surface and the long duplex DNA chain, the intercalated MB molecules are further

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prevented from reaching the electrode surface. Thus, with less free MB molecules present in solution, a

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significant electrochemical current drop is detected. By this “signal-off” mode, facile and sensitive

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homogenous electrochemical detection of miRNA is readily realized.

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Scheme 1. The principle of the label-free and enzyme-free homogeneous electrochemical strategy based on

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HCR amplification for miRNA assay.

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Feasibility Investigation of the miRNA Assay. The proof-of-concept experiments were carried

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out to investigate the feasibility of the proposed strategy for miRNA assay. First, the binding ability of MB

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toward different DNA conformations was tested. It has been reported that the diffusion current of MB

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bound with dsDNA decreases significantly as compared to that of ssDNA, and the diffusion current

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decrease is even more obvious when MB intercalates into G-quadruplex.23 As shown in Figure 1A, free MB

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in solution showed high electrochemical signal (curve a). A slight drop of the electrochemical signal was

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observed when MB was added in the solution containing the G-rich ssDNA (denoted as DNA1) in the

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absence of potassium ions (curve b), which may be due to the weak interaction between MB and DNA1

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that could not fold into G-quadruplex without potassium ions, thus the majority of MB were still in free

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state. When MB was added to the solution containing both DNA1 and DNA2, which are complementary to

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each other to form dsDNA, a significant drop of the electrochemical signal was detected (curve c). This

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could be the result of the intercalation of MB into the dsDNA, thus the diffusion current of MB in

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homogeneous solution was reduced due to a much lower apparent diffusion rate of the MB/dsDNA

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complex as compared to that of free MB. An even more significant drop was detected in the presence of

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MB, DNA1 and potassium ions (curve d), due to the fact that G-quadruplex was formed and the 10

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intercalation of MB into G-quadruplex further reduced the diffusion current of MB to the electrode surface.

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The aforementioned results were in accordance with the cyclic voltammetric (CV) results shown in Figure

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S1 (in Supporting Information), in which, under the same scan rate, the anodic peak currents of free MB

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(Figure S1A) were bigger than that of MB in the presence of dsDNA (Figure S1B), which in turn were

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bigger than that of MB in the presence of G-quadruplex (Figure S1C). Additionally, as shown in the insets

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of Figure S1, the anodic peak currents exhibited linear relationship with the square root of the scan rates

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(ν1/2) for the three systems, namely free MB, MB with dsDNA, and MB with G-quadruplex, suggesting

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typical diffusion-controlled processes. Therefore, the introduction of G-quadruplex to the reaction system

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can help to induce bigger electrochemical signal changes.

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The feasibility of the as-proposed label-free homogenous electrochemical miRNA assay was also

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investigated. As shown in Figure 1B, in the presence of MB, HP1 and HP2, a relatively large

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electrochemical signal was observed (curve a), indicating that the majority of MB were in free state. Once

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the target miRNA (let-7a) was present in the reaction system, if potassium ions were absent, the

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electrochemical signal showed an obvious drop (curve b), indicating the intercalation of MB into the

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duplex DNA chain formed via the target miRNA-triggered HCR; whereas, if potassium ions were present,

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the electrochemical signal further decreased (curve c), clearly demonstrating the occurrence of the target

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miRNA-triggered HCR and the subsequent formation of multiple G-quadruplex-incorporated long duplex

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DNA chain, with MB intercalated into which, the diffusion current of MB was significantly reduced.

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Furthermore, the formation of the HCR product was also proved by gel electrophoresis analysis (Figure S2

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in the Supporting Information). Thus, the aforementioned results demonstrated the feasibility of the

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as-proposed homogenous electrochemical approach for sensitive detection of miRNA.

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Moreover, the effect of the scan rate on the response of MB after HCR was investigated. As shown in 11

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Figure 1C, with the increase of the scan rate, the anodic potential (Epa) and the cathodic potential (Epc)

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shifted slightly toward the positive and the negative directions of potential, respectively. The anodic peak

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current (ipa) of MB was linearly proportional to the square root of the scan rate ranging from 0.01 to 0.3 V/s,

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and the regression equation was determined to be ipa= −1272.8ν1/2 + 107.5 (ipa, nA; ν, V/s) with a

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correlation coefficient of R2 = 0.9957, indicating that after HCR the rate-limiting step was still mass

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transport process through diffusion.

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Figure 1. (A) Differential pulse voltammograms of MB under different conditions: (a) MB; (b) MB +

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DNA1; (c) MB + dsDNA formed by DNA1 and DNA2; (d) MB + G-quadruplex formed by DNA1 in the

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presence of K+. (B) Differential pulse voltammograms under different conditions: (a) HP1 + HP2 + MB; (b)

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HP1 + HP2 + MB + let-7a; (c) HP1 + HP2 + MB + let-7a + K+. (C) Cyclic voltammograms of MB after

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HCR (with 20 pM let-7a present) at different scan rates: 0.01, 0.02, 0.05, 0.08, 0.1, 0.15, 0.2, 0.25 and 0.3

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V/s. Inset: the linear relationship between the anodic peak current and the square root of the scan rate. The

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concentrations of MB, DNA1, DNA2, HP1, HP2, and let-7a were 10 µM, 2 µM, 2 µM, 0.5 µM, 0.5 µM

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and 400 pM, respectively, if not otherwise stated.

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Optimization of Experimental Conditions. As illustrated in Scheme 1, in the presence of the

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target miRNA, G-quadruplex-incorporated duplex DNA chains are formed via the HCR between HP1 and

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HP2. To ensure high sensitivity of this biosensing strategy, sufficient amount of HP1 and HP2 are needed to

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form long DNA chains, so HP1 and HP2 with the same concentration of 0.5 µM were used in the

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experiments. In addition, to get the best performance of the proposed assay for miRNA, the MB

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concentration and the reaction time of this assay were investigated. As shown in Figure 2A, the DPV peak

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current change ∆ip (i.e. the difference between the DPV peak current in the presence of the target miRNA

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and that of the blank) exhibited a sharp increase as the MB concentration increased from 1 to 10 µM, but

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with the MB concentration further increased to 30 µM, ∆ip only showed a slight elevation. In our strategy,

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∆ip needs to be big enough to indicate the current change induced by the target, but too much MB in the 13

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reaction system may cause problems such as undesired adsorption. So 10 µM was chosen as the optimal

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MB concentration. As shown in Figure 2B, the DPV peak current decreased progressively with the reaction

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time increased from 0.5 to 2 h, but with the reaction time longer than 2 h the DPV peak current did not

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change much and leveled off. Therefore, 2 h were chosen as the optimal reaction time and used in the

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subsequent experiments.

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Figure 2. (A) The DPV peak current change (∆ip= ip − ip,0, in which ip is the DPV peak current in the

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presence of let-7a, and ip,0 is the DPV peak current of the blank) versus the MB concentration ranging from

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1 to 30 µM. The concentrations of HP1, HP2, and let-7a were 0.5 µM, 0.5 µM and 400 pM, respectively.

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(B) The relationship between the DPV peak current and the reaction time ranging from 0.5 to 3.0 h, in the

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presence of 10 µM MB, 0.5 µM HP1, 0.5 µM HP2, and 400 pM let-7a. The error bars represent the 14

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standard deviation of three repetitive measurements.

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Homogenous Electrochemical Detection of miRNA. Under the optimal experimental

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conditions, the target miRNA (let-7a) with different concentrations was added into the reaction system to

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evaluate the analytical performance of this biosensing platform. As illustrated in Figure 3, the DPV peak

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current gradually decreased with the increase of let-7a concentration from 0 to 800 pM, which was in

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accordance with the fact that miRNA with higher concentration would induce more HCR and more MB

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bound with the G-quadruplex-incorporated duplex DNA chain to reduce its diffusion current. The inset of

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Figure 3 showed a good linear correlation between the absolute value of DPV peak current and the

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logarithm of let-7a concentration ranging from 1 to 800 pM. The correlation equation was determined to be

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ip = 103.2lgC − 487.6 (ip, nA; C, pM) with a correlation coefficient of R2 = 0.9941, where ip was the DPV

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peak current and C was the let-7a concentration. The directly measured limit of detection for let-7a assay

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was 1 pM. In addition, the repeatability of the homogenous electrochemical biosensing method was further

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studied through 5 successive assays in the presence of 200 pM let-7a. The relative standard deviation (RSD)

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was determined to be 2.85%, indicating an acceptable repeatability for the as-proposed method for miRNA

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assay. These results demonstrated that sensitive detection of miRNA was indeed realized by the

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as-proposed label-free and enzyme-free homogenous electrochemical strategy, and this method should be

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sensitive enough to be adopted for the detection of miRNA in biological samples. More significantly, as

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compared to those labeling and/or immobilization-based electrochemical miRNA assays, the strategy we

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proposed here avoided the expensive labeling and sophisticated probe immobilization processes, making it

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cost effective and easy to implement.

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Figure 3. Differential pulse voltammograms corresponding to the analysis of let-7a with different

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concentrations: (a) 0, (b) 1, (c) 2, (d) 4, (e) 20, (f) 40, (g) 200, (h) 400 and (i) 800 pM. Inset: The linear

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relationship between the absolute value of the DPV peak current and the logarithm of let-7a concentration

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ranging from 1 to 800 pM. The error bars represent the standard deviation of three repetitive measurements.

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Selectivity of miRNA Assay. The selectivity of this experiment was further investigated by adding

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let-7a, let-7f and let-7g with the same concentration into the reaction system, respectively, in which HP1

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containing a perfectly complementary segment to let-7a was adopted. As shown in Figure 4, high DPV

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peak current was detected in the presence of let-7f or let-7g, which was slightly lower than that in the

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control experiment, whereas a significant drop of DPV peak current was observed when let-7a was present.

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The results demonstrated that the miRNA biosensing approach showed a high selectivity toward the target

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miRNA, even being able to distinguish one base mismatch. Thus, the as-proposed homogenous

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electrochemical miRNA assay exhibited high sequence specificity to discriminate target miRNA from its

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analogs in the same miRNA family.

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Figure 4. Comparison of the DPV peak currents in the presence of let-7a, let-7f and let-7g, respectively, in

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which “blank” indicates the condition in the absence of miRNAs. The concentrations of let-7a, let-7f and

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let-7g were all 400 pM. The error bars represent the standard deviation of three repetitive measurements.

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CONCLUSIONS

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In summary, we have developed a simple, label-free and enzyme-free homogeneous electrochemical

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biosensing strategy for sensitive detection of miRNA based on HCR signal amplification. In addition to

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good sensitivity for miRNA assay, this biosensing strategy exhibits excellent selectivity to distinguish even

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single-base mismatched miRNA. Furthermore, this method exhibits additional advantages of simplicity and

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low cost, since expensive labeling and sophisticated probe immobilization processes are avoided. Therefore,

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the as-proposed label-free and enzyme-free homogeneous electrochemical miRNA assay may become an

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alternative method for simple and sensitive miRNA detection and has great potential to be applied in

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miRNA-related clinical diagnostics and biochemical researches.

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Supporting Information

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Additional information including cyclic voltammograms of MB in different systems and the relationship

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between the peak current and the scan rate, as well as the gel electrophoresis results. This information is

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available free of charge via the Internet at http://pubs.acs.org/. 17

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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

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This work was funded by the National Natural Science Foundation of China (Nos. 21175076, 21375072

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and 21445002), the Open Foundation of State Key Laboratory of Electroanalytical Chemistry (No.

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SKLEAC201402), the Research Foundation for Distinguished Scholars of Qingdao Agricultural University

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(Nos. 663-1113311 and 663-1113320), and the Special Foundation for Taishan Scholar of Shandong

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Province.

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For TOC only

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