Label-Free and Enzyme-Free Homogeneous Electrochemical

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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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ACS Paragon Plus Environment


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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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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.

9



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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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220

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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

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

cost effective and easy to implement.

15



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272 273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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

285

analogs in the same miRNA family.

16


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286 287

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

288

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

289

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

290

CONCLUSIONS

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

miRNA-related clinical diagnostics and biochemical researches.

299

Supporting Information

300

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

301

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

302

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



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303 304 305

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

307

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

308

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

309

(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

313 314

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Label-Free and Enzyme-Free Homogeneous Electrochemical

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