Construction of a Cytosine-Adjusted Electrochemiluminescence

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Construction of a Cytosine-Adjusted Electrochemiluminescence Resonance Energy Transfer System for MicroRNA Detection Qiumei Feng, Mengying Wang, Xiaolei Zhao, and Po Wang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01829 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 3, 2018

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Construction of a Cytosine-Adjusted Electrochemiluminescence

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Resonance Energy Transfer System for MicroRNA Detection

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Qiumei Feng*, Mengying Wang, Xiaolei Zhao, and Po Wang*

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School of Chemistry and Materials Science, Jiangsu Normal University, Xuzhou

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221116, China

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* Corresponding authors.

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Tel.: +86 516 83403165. Fax: +86 516 83536977.

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E-mail: [email protected] (Q. Feng); [email protected] (P. Wang).

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ACS Paragon Plus Environment

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ABSTRACT

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The cytosines in cluster-nucleation sequences play a vital role in the formation of

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silver nanoclusters (Ag NCs). Here, an innovative electrochemiluminescence (ECL)

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resonance energy transfer (RET) sensing system was developed using CdS quantum

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dots (QDs) as ECL donor and Ag NCs as ECL acceptor. Modulation of the number of

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cytosines in the cluster-nucleation sequences allowed tuning of Ag NCs absorption

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bands to match with the ECL emission spectrum of CdS QDs, yielding effective

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ECL-RET. The sensitivity of detection was improved by dual target recycling

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amplification based on duplex-specific nuclease (DSN) and catalytic hairpin

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assembly. In the presence of target microRNA-21 (miRNA-21), DSN selectively

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cleaved the complementary DNA section (S1), resulting in the release of the

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transduction section (S2) and the reuse of miRNA-21 in the next recycling

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amplification. Interaction of the stem-loop structure of the DNA1 segment (H1) on

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CdS QDs-modified electrode with S2 led to the opening of the hairpin structure of H1

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and the formation of H1:S2 duplex. Then, hairpin DNA2 encapsulated Ag NCs

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hybridized with the remaining single-stranded DNA segment of H1, and the S2 strand

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was replaced. Finally, the dissociated S2 participated in subsequent reaction cycles,

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introducing Ag NCs to the electrode surface and leading to ECL signal quenching of

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the CdS QDs. The proposed sensor showed excellent performance in detecting

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miRNA-21 at a wide linear range from 1 fM to 100 pM. The practical application

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ability of the strategy was tested in HeLa cells with acceptable results, suggesting that

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the detection platform is a promising approach for disease diagnosis and molecular

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biology research.

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INTRODUCTION

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The use of electrochemiluminescence (ECL) for various bioanalytical applications has

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attracted increasing attention due to its high sensitivity, low background signal, wide

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dynamic response range, and not requiring external light source.1-4 Among various

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ECL emitters, CdS quantum dots (QDs) are attractive because of their high quantum

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yield, strong anti-interference capacity, simple film formation, and stable light

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emission.5,6 Because their physicochemical properties can be tailored by controlling

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the chemical composition or size to match wavelength requirements, CdS QDs are

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attractive as ECL resonance energy transfer (ECL-RET) donors.7,8 When the

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emission/absorption spectra of the donor/acceptor pair overlap, the energy of the

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donor is transferred to the acceptor, resulting in signal quenching in the donor or

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signal enhancement in the acceptor.9 Xu et al. developed interesting analytical

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systems based on QD ECL-RET, such as CdS:Mn QD-gold nanoparticles,10 CdS:Eu

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QD-gold nanorods,11 CdS QD-gold nanoparticle dimers,12 CdS QD-silver

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nanoclusters,13 and CdTe QD-gold nanodendrites.14 These strategies offer precise

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quantitative detection by normalizing environmental variations, such as false-positive

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signal, background light, and scattering. Efficient ECL-RET depends critically on

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perfect energy overlapping of the donor–acceptor pair.15,16 Hence, the identification of

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an acceptor with a UV–vis absorption spectrum matching the ECL energy spectra of

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CdS QDs is important for the construction of ECL-RET sensor.

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Because of the strong quantum confinement effect of free electrons in the

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ultrasmall size regime, silver nanoclusters (Ag NCs) have certain advantages: they are

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non-toxic, biocompatible, and have good physical, optical, and electronic

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properties.17-19 In general, Ag NCs are synthesized by reducing Ag ions using

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chemical reductants or light irradiation, which cause Ag NCs to aggregate and form 3


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large nanoparticles.20 To overcome this problem, specific functional oligonucleotide

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(denoted as cluster-nucleation) sequences are used to modify Ag NCs.21,22 For

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example, cytosine-rich DNA sequences are used as templates to synthesize Ag NCs

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with increased stability.23 The position and number of cytosines in the

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cluster-nucleation sequence play a vital role in the formation of Ag NC species. Yeh

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et al. showed that the emission spectra of Ag NCs can be tuned by sliding one of the

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polycytosine heads with respect to the enhancer cluster-nucleation sequence.24

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Therefore, the absorption bands of Ag NCs can be tuned by adjusting the number of

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cytosines to match the better acceptor with an absorption band that overlaps with the

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ECL emission spectra of CdS QDs.

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Sensitive and selective detection of microRNA (miRNA) is highly significant for

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clinical diagnosis and treatment.25,26 The ultrasensitive determination of miRNA

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remains a challenge due to their small size and low abundance in cancer cells.27-30 In

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recent years, researchers have explored various amplification strategies for miRNA

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detection, such as target-catalyzed hairpin assembly,31 rolling circle amplification

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(RCA),32 polymerase chain reaction (PCR),33 strand displacement amplification

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(SDA),34

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amplification methods have been used to amplify targets, probes, or signals. Among

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them, the recycling amplification of targets is one of the most promising target

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regeneration approaches, in which one target oligonucleotide can lead to multiple

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binding events. For example, duplex-specific nuclease (DSN)-assisted amplification

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allows recognition of the probe-miRNA duplexes and selective cleavage of the signal

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probe. As a result, the released target miRNA is available for subsequent

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hybridization, decreasing the detection limit and increasing sensitivity.35 Recently, our

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group achieved detection of miRNA-155 at the femtomolar level using fluorescence

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quenching of gold nanoparticles to Ag NCs.36 However, certain nucleic acid strands

and

nuclease-assisted

multiple

amplification.27

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These

recycling

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were produced in the homogeneous solution that could not be easily separated from

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the mixture, leading to a high background signal during testing. Hence, the

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development of novel testing conditions on a macroscopic surface would be desirable

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to fully exploit the potential of the method for real-world applications.

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Herein, an ECL-RET sensor coupled with a dual target recycling amplification

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strategy was designed based on the efficient CdS QDs-Ag NCs RET pair. In this

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design, the stem-loop structure DNA2 segment (H2) consisted of a hairpin DNA and a

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cluster-nucleation sequence at the 3′ end. The addition of a reducing agent and Ag

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cations yielded H2-encapsulated Ag NCs (H2-Ag NCs). Systematic alteration of the

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number of cytosines in the cluster-nucleation sequence changed the UV–vis

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absorption peaks of Ag NCs in a periodic fashion to 495 nm (C5-5), 520 nm (C6-6), 528

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nm (C7-7), and 533 nm (C8-8). Because of the complete overlap of spectra, C6-6 Ag

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NCs were the most effective quenchers for the ECL emission of CdS QDs, with an

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emission spectrum centered at 520 nm. Coupled with dual target recycling

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amplification, an ultrasensitive ECL-RET sensor was developed for the detection of

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miRNA-21 with a detection limit of 600 aM.

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EXPERIMENTAL SECTION

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Reagents. Potassium peroxydisulfate (K2S2O8), sodium sulfide (Na2S·9H2O), and

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cadmium nitrate tetrahydrate (Cd(NO3)2·4H2O) were supplied by Sinopharm

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Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium

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(DMEM), fetal bovine serum, silver nitrate (AgNO3), tris (2-carboxyethyl) phosphine

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hydrochloride (TCEP), sodium borohydride (NaBH4), N-hydroxysuccinimide (NHS),

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N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC), bovine

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serum albumin (BSA), and 6-mercapto-1-hexanol (MCH) were bought from 5


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Sigma-Aldrich (St. Louis, MO, USA), and used without further purification. DSN was

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purchased from Evrogen (Russia), and DSN buffer consisted of 1 mM DTT, 50 mM

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Tris-HCl, and 5 mM MgCl2. Carboxylated magnetic beads (20 nm, MB) were

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obtained from Xi’an GoldMag Nanobiotech Co., Ltd. (Xi’an, China). The 0.1 M

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K2S2O8 prepared by a phosphate buffered solution (PBS, 0.1 M KH2PO4–Na2HPO4,

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pH 8.3) was used as the coreactant for ECL detection. Ultrapure water was obtained

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using a Millipore water purification system (resistivity ≥18.2 MΩ·cm). All

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oligonucleotides were ordered from Sangon Biological Engineering Technology &

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Services Co., Ltd. (Shanghai, China), and purified using high-performance liquid

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chromatography. Oligonucleotide sequences are listed in Table 1.

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Apparatus. All the ECL emission measurements were performed on an MPI-A

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ECL analyzer (Xi’an Remax Electronic Science & Technology Co., Ltd., Xi’an,

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China, 350–650 nm) at room temperature. For detection, the voltage of the

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photomultiplier tube (PTM) was set to −700 V. The potential scan was carried out

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from 0 to −1.05 V at a scan rate of 100 mV s−1. The electrochemical cell consisted of

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a traditional three-electrode system with a modified glassy carbon working electrode

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(GCE), a silver/silver chloride reference electrode, and a Pt wire counter electrode.

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The UV–vis absorption spectra of the synthesized Ag NCs were acquired with a

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Shimadzu UV-3600 UV–vis–NIR spectrophotometer (Shimadzu Co., Japan).

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Transmission electron microscopy (TEM) images of Ag NCs were performed on a

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JEOL 2000 instrument operating at an accelerating voltage of 200 kV. Atomic force

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microscopy (AFM) images were carried out with a cross-sectional atomic force

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microscopy (Agilent Technologies, Inc. USA) in tapping mode. The hydrodynamic

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diameters of Ag NCs were measured by dynamic light scattering (DLS) on a 90 6


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Plus/BI-MAS

equipment

(Brookhaven,

USA).

Electrochemical

impedance

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spectroscopy (EIS) measurements of different modified electrodes were performed on

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a PGSTAT30/FRA2 system (Autolab, the Netherlands) with the following

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experimental parameters: DC potential, 0.21 V; frequency range, 104–0.1 Hz; and

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amplitude, 0.01 V. Gel electrophoresis analysis of DNA catalytic hairpin assembly

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amplification was performed on a Bio-Rad electrophoresis analyzer (USA) and

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imaged using the Bio-Rad ChemDoc XRS (USA).

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Synthesis of CdS QDs. In brief, 0.1861 g Cd(NO3)2·4H2O was added to 30 mL of

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ultrapure water and heated to 70 °C under constant magnetic stirring. Then, a fresh

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Na2S·9H2O solution (83 mM) in 30 mL ultrapure water was slowly dropped into the

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above solution, and orange–yellow precipitates were instantly obtained. The stirring

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reaction was maintained for 3 h at 70 °C, and the obtained precipitates were

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centrifuged and thoroughly washed with absolute ethanol and ultrapure water. Finally,

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after the centrifugation of the precipitate in ultrapure water at 9000 rpm for 10 min,

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CdS QDs at a concentration of 2.2 mg mL−1 were retrieved from the upper yellow

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colloidal solution.

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Formation of H2-Ag NCs. H2-encapsulated Ag NCs were synthesized according

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to the literature with some modifications.37 Initially, 2 µL of 9 mM AgNO3 solution

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was quickly added to 170 µL of 15 µM template H2 strand (C6-6, prepared in 10 mM

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Tris-HCl buffer, pH 6.6). The mixed solution was vigorously vortexed for 5 s and

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centrifuged at 8000 rpm for 30 s. The solution was incubated in the dark for 30 min at

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4 °C. For H2-Ag NCs, 2 µL of freshly prepared 9 mM NaBH4 solution was added,

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and the obtained mixture was vortexed again and centrifuged. Finally, the resulting

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pale yellow mixture was stored in the dark for 2 h at 4 °C, and H2-Ag NCs (number

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of cytosines in the cluster-nucleation sequence: C6-C6) were obtained. Various

7


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cytosine-adjusted H2-Ag NCs (number of cytosines in the cluster-nucleation

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sequence: C5-C5, C7-C7, and C8-C8) were synthesized using the same procedure.

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Fabrication of S2. The S2 strand was obtained by DSN-assisted target recycling

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amplification. Scheme 1B illustrates the preparation process of S2. Firstly, a mixed

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solution containing EDC (20 mg mL−1) and NHS (10 mg mL−1) was added to a

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carboxylated magnetic bead (MB) suspension, and the resultant mixture was

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incubated at 37 °C for 60 min. After rinsing with PBS, 200 µL of amino-modified

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S1:S2 strand (2 µM) was injected into the carboxyl group-activated MB and incubated

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for 60 min with gentle shaking, resulting in the formation of MB-S1:S2

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bioconjugates. Then, 2 wt.% BSA was used to block the nonspecific active binding

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sites of the MB. After washing, the acquired MB-S1:S2 conjugate was resuspended in

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20 mM PBS containing 5 mM Mg2+, and different concentrations of miRNA-21 were

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added, followed by incubation at 37 °C for 60 min. Next, 0.1 U of DSN (dissolved in

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25 mM Tris-HCl and 50% glycerol) was added and incubated at 37 °C for 70 min.

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Finally, DSN stop solution containing 5 mM EDTA was introduced to deactivate the

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DSN, and the special solution containing the S2 strand was removed by magnetic

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force.

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Gel Electrophoresis. The catalytic hairpin assembly amplification was confirmed

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by polyacrylamide gel electrophoresis. Firstly, sample 1 and sample 2 were prepared

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by incubating the mixture of H1 and S2 and the mixture of H2, H1, and S2 at 37 °C

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for 80 min. Then, gel electrophoresis was performed by adding 7 µL of different DNA

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structures (S2, H1, H2, sample 1, and sample 2) to the mixture of 1.5 µL loading

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buffer and 1.5 µL UltraPower TM dye. Then, these mixtures were injected into a 10%

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native polyacrylamide gel (prepared with 5 × TBE buffer), and electrophoresis was

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run at 100 V for 60 min. Finally, the resulting board was illuminated with UV light

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and photographed using Molecular Imager Gel Doc XR. 8


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Preparation of Cellular Extracts. Prior to analysis, total RNA containing

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miRNA-21 was extracted from human cervical cells (HeLa cells) according to a

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published method.38 In a typical procedure, HeLa cells were seeded in DMEM

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supplemented with penicillin (100 µg mL−1), streptomycin (100 µg mL−1), and 10%

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(v/v) fetal calf serum and maintained at 37 °C in a humidified atmosphere (5% CO2

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and 95% air). HeLa cells were collected at the exponential phase of growth and rinsed

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with PBS three times. After detachment from culture flasks using trypsin and

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inactivation with fetal calf serum, the number of HeLa cells was counted using a

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hemocytometer. Then, in the RNase-free environment, HeLa cells were lysed with

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RNA extraction reagent for complete dissociation of the nucleoprotein complex. Next,

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chloroform was added to the system, and the mixture was vortexed for 30 s, followed

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by centrifugation at 12000 rpm for 15 min at 4 °C. The upper aqueous phase was

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placed into a new tube, and isopropanol was added to precipitate the RNA. After

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centrifugation and rinsing with 75% ethanol, the RNA was dissolved in RNase-free

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water for further detection.

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Construction of the ECL-RET Sensor. Scheme 1C is a schematic representation

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of the stepwise fabrication process of the ECL-RET sensor. Before surface

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modification, a GCE was sequentially polished with 1.0, 0.3, and 0.05 µm alumina

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powder, followed by sonication in anhydrous ethanol and ultrapure water for 3 min.

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The cleaned GCE was firstly coated with 10 µL of CdS QDs solution and evaporated

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in air at room temperature, yielding CdS QDs/GCE. Subsequently, the modified

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electrode was incubated with 100 µL of H1 solution at 4 °C overnight to anchor H1 to

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the electrode surface (H1/CdS QDs/GCE). Hairpin DNA strands (H1 and H2) were

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annealed by heating at 95 °C for 5 min, and then allowed to cool to room temperature

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before use. The thiolated H1 was activated using 10 mM TCEP for 1 h to cleave the

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S–S bonds, and the excess TCEP was removed with a Millipore centrifugal filter (10 9


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kDa). After rinsing with 20 mM PBS containing 5 mM Mg2+, 1 mM MCH solution

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was added to block the nonspecific active binding sites of CdS QDs. The resultant

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MCH/H1/CdS QDs/GCE was further incubated with a special solution containing the

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S2 strand from DSN-assisted target recycling amplification.

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Analytical Procedure. The prepared S2/MCH/H1/CdS QDs/GCE was further

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incubated in H2-Ag NCs solution. After washing with 20 mM PBS containing 5 mM

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Mg2+, Ag NCs were introduced onto the modified electrode surface. Finally, the fully

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constructed ECL sensor was placed in 4 mL of 0.1 M PBS (pH 8.3) containing 0.1 M

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K2S2O8 for ECL detection.

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RESULTS AND DISCUSSION

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Construction of the ECL-RET Sensor. Scheme 1 depicts the working principle of

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the sensor, including the formation of H2-Ag NCs with various cluster-nucleation

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sequence design strategies, the DSN-assisted target recycling amplification, the

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construction of the ECL sensing platform with the catalytic hairpin assembly, and the

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RET response from the quenching of Ag NCs. Briefly, in the DSN-assisted target

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recycling system as the first amplification step (Scheme 1B), the S1:S2 strand

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consisted of the recognition section (S1) and the transduction section (S2). S1 was

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complementary to miRNA-21, and S2 triggered the catalytic hairpin assembly as the

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second recycling amplification step on the electrode surface. Firstly, the S1:S2 strand

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with an amino group at the 5′ end of S1 was immobilized onto the surface of carboxyl

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group-activated MB. Then, in the presence of target miRNA-21, DNA-RNA

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heteroduplexes between S1 and miRNA-21 were formed as the DSN substrate. As a

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duplex-specific nuclease, DSN selectively cleaves double-stranded DNA or DNA in

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the DNA–RNA heteroduplex, whereas it has no activity against single-stranded DNA

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or RNA. Hence, the S1 strand in DNA–RNA heteroduplexes was selectively cleaved, 10


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resulting in the release of miRNA-21 and the dissociation of the S2 strand from MB.

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Since miRNA-21 remained intact during the cleavage process, the released

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miRNA-21 could be reused in subsequent reaction cycles. Eventually, one miRNA-21

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could lead to the release of numerous S2 strands, triggering the second recycling

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amplification on the electrode. As shown in Scheme 1C, H1 labelled with a thiol at its

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5′ end was constructed on a GCE modified with CdS QDs as the recognition probe.

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Interaction of the immobilized H1 with the S2 strand resulted in unfolding of the

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hairpin structure of H1 and modification to a rod-like structure through

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complementary hybridization, leading to the formation of the H1:S2 duplex. H2-Ag

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NCs hybridized with the remaining single-stranded DNA segment of H1, resulting in

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the formation of H1:S2:H2-Ag NCs as an intermediate. Then, the S2 strand was

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replaced by quick rearrangement, and the H1:H2-Ag NCs duplex was generated on

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the electrode. The dissociated S2 was reused in subsequent reaction cycles.

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Ultimately, after sufficient amplification, many Ag NCs were introduced onto the

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electrode surface, and the fabricated sensor was immersed in PBS containing K2S2O8

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for ECL detection. In the absence of target miRNA-21, the DSN-assisted target

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recycling system as the first amplification was negative, and the amount of Ag NCs

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formed according to the quantity of S2 strands on the electrode was restricted. Hence,

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the amount of assembled Ag NCs strongly depended on the concentration of target

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miRNA-21.

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In the CdS QDs-K2S2O8 ECL system, as the electrode potential became negative

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enough, CdS QDs on the electrode were reduced to (CdS)•− radicals by charge

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injection. Meanwhile, the coreactant S2O82− was reduced to the strong oxidant SO4•−,

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converting (CdS)•− to the excited state (CdS)*. As it returned to the ground state,

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(CdS)* emitted light, and a strong ECL signal was obtained. The TEM image of CdS

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QDs was shown in Figure 1A. The average size of the synthesized CdS QDs was 11


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about 6 nm. The ECL spectrum of the CdS QDs modified GCE exhibited a strong

262

ECL emission centered at 520 nm (Figure 1B). To generate an effective ECL-RET

263

sensor, it was essential to find a good energy acceptor for CdS QDs with an

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absorption band possessing a perfect spectral overlap with the ECL emission

265

spectrum of the CdS QDs.

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Characteristics of H2-Ag NCs. Ag NCs consisting of several to tens of atoms

267

smaller than 2 nm can be synthesized using specific cytosine-rich single stranded

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oligonucleotide sequence as the template and NaBH4 as the reducing agent for the

269

reduction of Ag cations.39 Such Ag NCs possessed an absorption band at a peak of

270

492 nm in the UV–vis spectrum.40 The number of cytosines in the cluster-nucleation

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sequence has an important impact on the position of the absorption band for Ag NCs.

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To test this hypothesis, we showed various cluster-nucleation sequence design

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strategies in Scheme 1A. C-series represented the size of the polycytosine heads,

274

which were altered in the cluster-nucleation sequences (see Table 1). Thus, C5-5

275

represented a C-rich cluster-nucleation sequence designed with two C5 polycytosine

276

heads at the 3′ and 5′ ends. These two C5 polycytosine heads were separated by the

277

TTAAT-linker. Except for the changes in the number of cytosines, C6-6, C7-7, and C8-8

278

showed the same design. The length of the polycytosine head, which interacted with

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the Ag cations, affected the UV–vis absorption spectra of the template silver clusters.

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As depicted in Figure 2A, an increase in the length of the polycytosine heads shifted

281

the peak of the absorption spectrum to red (C5-5, 495 nm; C6-6, 520 nm; C7-7, 528 nm;

282

and C8-8, 533 nm). It was likely that different sizes of polycytosine heads stabilized

283

different silver cluster species. The absorption band of C6-6 Ag NCs (520 nm) showed

284

complete overlap with the ECL emission spectrum of CdS QDs (520 nm, Figure 1B),

285

which was highly important for efficient energy transfer.14 The greater the overlap

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appeared between the donor ECL spectrum and the acceptor absorption spectrum at a 12


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short separation distance, the greater the energy transfer efficiency of the ECL-RET

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system occurred. Thus, C6-6 H2-Ag NCs were selected as the quencher for the ECL

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emission of CdS QDs.

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Figure 2B shows the typical absorption spectrum of C6-6 H2-Ag NCs. Except for

291

the absorption band at 520 nm assigned to ultrasmall Ag NCs, another strong

292

absorption peak centered at 395 nm was clearly observed, which corresponded to the

293

larger Ag nanoparticles in aqueous solution. To further characterize the prepared C6-6

294

H2-Ag NCs, the morphology was examined by TEM. Figure 2C showed that the Ag

295

NCs exhibited well dispersion status, and the average size was about 2 nm. In order to

296

investigate the microscopic property of Ag NCs, the HR-TEM image of the profile of

297

a single Ag NC was presented in the insert of Figure 2C. The lattice fringe spacing of

298

the crystal was measured to be 0.22 nm, corresponding to the Ag (102) lattice based

299

on JCPDS card No. 87-0598. Moreover, AFM technique was employed to

300

characterize the sensing system. The bare GCE exhibited a smooth and flat surface

301

(Figure 2D), while multilayer spheroidal particles were clearly observed after the

302

assembly of CdS QDs on GCE (Figure 2E). In Figure 2F, many smaller particles

303

highlighted in green circles were observed on the surface of CdS QDs after the

304

incubation of S2/MCH/H1/CdS QDs/GCE with H2-Ag NCs solution, demonstrating

305

the successful introduction of Ag NCs onto the sensing interface.

306

Characteristics of the ECL-RET Sensor. The feasibility of catalytic hairpin

307

assembly was confirmed by gel electrophoresis. In Figure 3A, lanes 1–3 showed the

308

S2, H1, and H2 bands, respectively. The H1:S2 complex band, which was shown in

309

lane 4, had a larger molecular weight than both S2 (lane 1) and H1 (lane 2). After the

310

addition of H2 to the H1:S2 complex, the bands corresponding to the mixture of H1

311

and S2 faded, and a strong band was observed in lane 5. The band corresponding to

13


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312

the S2 strand became bright again, demonstrating the successful interaction of H2

313

with H1 and the release of the S2 strand.

314

To efficiently monitor the interfacial properties of the modified electrodes, the ECL

315

responses were used to explore the assembly processes of the ECL sensor step by step

316

in Figure 3B. Construction of CdS QDs on the bare GCE resulted in the appearance of

317

a distinct ECL signal (curve a). In the presence of coreactant K2S2O8, the CdS QDs

318

acted as an ECL emitter and generated an ECL peak at −1.001 V. The successive

319

introduction of H1 (curve b), MCH (curve c), and S2 (curve d) resulted in a

320

progressive decrease in the ECL signal. This phenomenon was attributed to the

321

formation of a lower conductivity layer on the surface of the electrode. However,

322

hybridization of H2-Ag NCs generated an obvious quenching ECL signal (curve e),

323

indicating that Ag NCs were suitable ECL energy acceptors to effectively quench

324

ECL from CdS QDs. On the other hand, Ag NCs also catalyzed the reduction of

325

coreactant S2O82− to SO42−, decreasing the generation of the SO4•− radical and the

326

ECL signal.13

327

The step by step assembly of the modified electrode was also monitored by EIS,

328

which is a simple but powerful electrochemical tool. In the EIS Nyquist plots, the

329

semicircle portion diameter corresponded to the electron-transfer resistance (Ret). As

330

shown in Figure 3C, the original bare GCE exhibited a very small Ret (curve a),

331

indicating good electrical conductivity of the interface. Compared with the bare GCE,

332

the Ret increased after the immobilization of CdS QDs on the electrode surface (curve

333

b). After the subsequent self-assembly of H1, MCH, and S2, the Ret increased

334

gradually as the experiment proceeded (curves c–e), reflecting the successful

335

assembly of the sensing system. The reason for the increase in resistance was that the

336

assembled nonconductor progressively obstructed the mass transport and electron

337

transfer of the electrochemical probe to the electrode surface by promoting the 14


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hindrance effect. Incubation with H2-Ag NCs decreased the Ret (curve f), suggesting

339

that the Ag NCs accelerated the electron transfer despite the additional negative

340

charges introduced onto the electrode surface through nucleic acid hybridization.

341

Taken together, the ECL responses and EIS measurements demonstrated the

342

successful preparation of the proposed sensor.

343

Analytical Performance. To achieve the best sensing performance, the

344

experimental conditions affecting the ECL intensity were optimized. Figure 4A

345

showed the effect of DSN nicking time on the ECL response of the sensor. Increasing

346

the incubation time with the DSN resulted in a gradual decrease of the ECL signal

347

from 20 min to 70 min, and a plateau was reached after 70 min, demonstrating that 70

348

min was sufficient for the nicking process. The hybridization time between H1 and

349

H2-Ag NCs was also investigated in Figure 4B. Because of the increase in Ag NCs on

350

the electrode surface, the ECL intensity slowly decreased and tended to level off with

351

hybridization times above 80 min. Thus, 70 min of nicking time for the DSN and 80

352

min hybridization time between H1 and H2-Ag NCs were selected as the optimal

353

incubation conditions for subsequent assays.

354

Under the optimized experimental conditions, the potential applications and the

355

sensitivity of the ECL-RET sensor were assessed through incubation with different

356

concentrations of miRNA-21. As shown in Figure 4C, the ECL signal gradually

357

decreased with increasing miRNA-21 concentrations. The ECL increment ∆I (∆I = I0

358

– I, where I0 and I were ECL signals of S2/MCH/H1/CdS QDs/GCE before and after

359

hybridization with H2-Ag NCs, respectively) was logarithmically related to the

360

concentration of miRNA-21 in the range from 1 fM to 100 pM (Figure 4D). A

361

correlation coefficient was obtained to be 0.992, and the detection limit corresponding

362

to a signal-to-noise ratio of 3 was 600 aM. The analytical parameters of the developed

363

sensing platform including linear range and detection limit were compared with 15


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364

various reference methods. As shown in Table 2, the detection performances of the

365

proposed system were comparable with most reference methods,41-52 indicating that

366

the sensing system provided a promising platform for miRNA-21 detection.

367

The stability of the sensor was evaluated by determining the consecutive ECL

368

responses for 12 cycles as shown in Figure 5A. The ECL intensities were similar and

369

consistent through all the cycles, demonstrating the stability of the ECL-RET sensor.

370

The reproducibility was estimated by analyzing the ECL responses of 10 pM

371

miRNA-21 using eight independent electrodes under the same conditions. A relative

372

standard deviation (RSD) of 4.6% was obtained, showing an acceptable

373

reproducibility for miRNA-21 assay. The long-term storage stability was explored by

374

continuous detection with the same sensor, which was stored at 4 °C in the dark when

375

not in use. ECL measurements were performed for 15 days at 3-day intervals. As

376

shown in Figure 5B, no obvious change was observed during the initial 9 days,

377

indicating that the as-prepared ECL-RET sensor exhibited excellent stability for

378

storage within 9 days. However, the ECL increment ∆I gradually declined beyond 9

379

days, which was attributed to the structure change of Ag NCs.

380

One important challenge associated with the miRNA assay was the ability to

381

distinguish the target miRNA from family members with similar length and

382

sequence.53 The selectivity of the proposed sensor was therefore investigated by

383

measuring the ECL responses to a series of RNA sequences, including mutated

384

sequences

385

miRNA-21) and other miRNAs (miRNA-141, miRNA-155, miRNA-182, and

386

miRNA-143). As depicted in Figure 5C, in the presence of complementary target

387

miRNA-21 (a), the ECL increment ∆I was greater than those of single-base

388

mismatched miRNA-21 (b), three-base mismatched miRNA-21 (c), miRNA-141 (d),

389

miRNA-155 (e), miRNA 182 (f), and miRNA-143 (g). Moreover, the cross-selectivity

(single-base

mismatched

miRNA-21

and

16


three-base mismatched

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390

of the proposed system was tested by detection of target miRNA-21 in the presence of

391

interferents, including single-base mismatched miRNA-21, three-base mismatched

392

miRNA-21, miRNA-141, miRNA-155, miRNA-182, and miRNA-143. It was

393

demonstrated that the coexistence of miRNA-21 with these control sequences (h) did

394

not cause a distinct signal change compared with that of miRNA-21 alone (a),

395

indicating high selectivity of the system for miRNA-21 analysis, which was attributed

396

to the fact that only the perfectly matched miRNA-21 was capable of triggering the

397

DSN-assisted dual-signal amplification strategy.

398

To validate the practical applicability of this designed sensor for the quantitative

399

assay of miRNA-21 in a complex biological matrix, the concentration of miRNA-21

400

in HeLa cell extracts was measured. The average copy number of miRNA-21 in a

401

single cell was approximately 2.16 × 103, which was in good agreement with the

402

reported results of miRNA-21 expression.39,54,55 In addition, recovery testing was

403

performed by spiking 10 pM miRNA-21 into 50-fold diluted normal human serum.

404

The resulting recovery was 106%, demonstrating that the ECL-RET sensor has

405

potential applicability in real biological samples.

406

CONCLUSIONS

407

In summary, a promising platform was explored for the ultrasensitive detection of

408

miRNA-21 based on the construction of a cytosine-adjusted ECL-RET system

409

coupled with a dual signal amplification strategy. The proposed system exhibited

410

significant ECL-RET responses for target analysis, which were obtained by

411

optimizing Ag NC species through regulating the number of cytosines in the

412

cluster-nucleation sequences. DSN-assisted signal amplification and catalytic hairpin

413

assembly were used for the highly sensitive detection of miRNA-21 with a low

414

detection limit of 600 aM, paving the way for the assay of real samples with low 17


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415

miRNA abundance. In addition to its high sensitivity, the ECL-RET sensing platform

416

showed good performances for miRNA-21 detection, including low cost, ease of

417

operation, good stability and reproducibility, and high selectivity. The use of the

418

proposed system for the detection of miRNA-21 in HeLa cell extracts and human

419

serum samples exhibited acceptable results, suggesting potential applicability of the

420

sensing platform in early cancer diagnosis and molecular biology research.

421

AUTHOR INFORMATION

422

Corresponding Authors

423

Tel.: +86 516 83403165. Fax: +86 516 83536977.

424

E-mail: [email protected] (Q. Feng); [email protected] (P. Wang).

425

Author Contributions

426

All authors have given approval to the final version of the manuscript.

427

Notes

428

The authors declare no competing financial interest.

429

ACKNOWLEDGMENTS

430

This work was supported by the National Natural Science Foundation of China (Nos.

431

21705062, 21675067), the Natural Science Foundation of Jiangsu Province

432

(BK20170228), the Natural Science Foundation of Jiangsu Normal University

433

(16XLR012), the State Key Laboratory of Analytical Chemistry for Life Science, and

434

the project funded by the Priority Academic Program Development of Jiangsu Higher

435

Education Institutions.

436

18


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(48) Wang, M.; Yin, H.; Shen, N.; Xu, Z.; Sun, B.; Ai, S. Signal-on Photoelectrochemical

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Biosensor for MicroRNA Detection Based on Bi2S3 Nanorods and Enzymatic Amplification.

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Biosens. Bioelectron. 2014, 53, 232–237.

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(49) Wei, X.; Duan, X.; Zhou, X.; Wu, J.; Xu, H.; Min, X.; Ding, S. A Highly Sensitive

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SPRi Biosensing Strategy for Simultaneous Detection of Multiplex miRNAs Based on Strand

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Displacement Amplification and AuNP Signal Enhancement. Analyst 2018, 143, 3134–3140.

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(50) Zhang, J.; Zhang, W.; Gu, Y. Enzyme-Free Isothermal Target-Recycled Amplification

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Combined with PAGE for Direct Detection of MicroRNA-21. Anal. Biochem. 2018, 550,

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117–122.

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(51) Chen, A.; Ma, S.; Zhuo, Y.; Chai, Y.; Yuan, R. In Situ Electrochemical Generation of

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Electrochemiluminescent Silver Naonoclusters on Target-Cycling Synchronized Rolling

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Circle Amplification Platform for MicroRNA Detection. Anal. Chem. 2016, 88, 3203–3210.

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(52) Feng, X.; Gan, N.; Zhang, H.; Li, T.; Cao, Y.; Hu, F.; Jiang, Q. Ratiometric Biosensor

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Array for Multiplexed Detection of MicroRNAs Based on Electrochemiluminescence

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Coupled with Cyclic Voltammetry. Biosens. Bioelectron. 2016, 75, 308–314.

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(53) Moltzahn, F.; Olshen, A. B.; Baehner, L.; Peek, A.; Fong, L.; Stoppler, H.; Simko, J.;

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Hilton, J. F.; Carroll, P.; Blelloch, R. Microfluidic-Based Multiplex qRT-PCR Identifies

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Diagnostic and Prognostic MicroRNA Signatures in the Sera of Prostate Cancer Patients.

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Cancer Res. 2011, 71, 550–560.

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(54) Feng, Q. M.; Shen, Y. Z.; Li, M. X.; Zhang, Z. L.; Zhao, W.; Xu, J. J.; Chen, H. Y.

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Dual-Wavelength Electrochemiluminescence Ratiometry Based on Resonance Energy

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Transfer between Au Nanoparticles Functionalized g-C3N4 Nanosheet and Ru(bpy)32+ for

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MicroRNA Detection. Anal. Chem. 2016, 88, 937–944.

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(55) Meister, G.; Landthaler, M.; Patkaniowska, A.; Dorsett, Y.; Teng, G.; Tuschl, T.

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Human Argonaute2 Mediates RNA Cleavage Targeted by miRNAs and siRNAs. Mol. Cell

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2004, 15, 185–197.

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606

FIGURE CAPTIONS

607

Scheme 1. Schematic illustration of the cytosine-adjusted ECL-RET sensor based on

608

a dual recycling amplification strategy for the detection of miRNA-21. (A) Synthesis

609

of C-series H2-Ag NCs. (B) Preparation of S2 based on DSN-assisted target recycling

610

amplification. (C) Construction of the ECL-RET sensor with the catalytic hairpin

611

assembly amplification.

612

Figure 1. (A) TEM image of the as-prepared CdS QDs. (B) ECL spectrum intensities

613

of CdS QDs obtained using a series of optical filters (460, 480, 500, 520, 540, 560,

614

580, 600, 620, and 640 nm).

615

Figure 2. (A) UV–vis absorption spectra of C-series H2-Ag NCs. (B) UV–vis

616

absorption spectrum of C6-6 H2-Ag NCs. (C) TEM image and HR-TEM image (insert)

617

of C6-6 H2-Ag NCs. (D) AFM image of bare GCE. (E) AFM image of CdS QDs

618

modified GCE. (F) AFM image of S2/MCH/H1/CdS QDs/GCE incubated with

619

H2-Ag NCs solution.

620

Figure 3. (A) Gel electrophoresis analysis of DNA catalytic hairpin assembly

621

amplification: (lane 1) S2, (lane 2) H1, (lane 3) H2, (lane 4) the mixture of H1 and S2,

622

(lane 5) the mixture of H1, S2, and H2. In lanes 1–5, the concentrations of all

623

sequences were 10 µM. (B) Cyclic ECL intensities on potential curves of (a) CdS

624

QDs/GCE,

625

S2/MCH/H1/CdS QDs/GCE, (e) S2/MCH/H1/CdS QDs/GCE further incubated with

626

H2-Ag NCs solutions. (C) EIS of the GCE at different stages: (a) bare GCE, (b) CdS

627

QDs/GCE, (c) H1/CdS QDs/GCE, (d) MCH/H1/CdS QDs/GCE, (e) S2/MCH/H1/CdS

628

QDs/GCE, (f) S2/MCH/H1/CdS QDs/GCE further incubated with H2-Ag NCs

629

solutions.

(b)

H1/CdS

QDs/GCE,

(c)

MCH/H1/CdS

25


QDs/GCE,

(d)

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

630

Figure 4. (A) Effect of DSN reaction time on the ECL response of the sensor. (B)

631

Effect of hybridization time between H1 and H2-Ag NCs on the ECL response of the

632

sensor. (C) ECL-time curves for the detection of different concentrations of

633

miRNA-21 (from 0 fM to 100000 fM). (D) The resulting linear relationship between

634

the relative ECL intensity (∆I) and the logarithm of miRNA-21 concentration. Five

635

independent measurements were performed to determine the standard deviation.

636

Figure 5. (A) ECL emission from the H2-Ag NCs/S2/MCH/H1/CdS QDs/GCE

637

sensor with miRNA-21 at the concentration of 10 pM under continuous cyclic

638

potential scans for 12 cycles. (B) Bar graph of the long-term storage stability of the

639

ECL-RET sensor (0–15 days). (C) Bar graph of ∆I for the ECL-RET sensor after

640

incubation with different RNA sequences (10 pM): (a) target miRNA-21, (b)

641

single-base mismatched miRNA-21, (c) three-base mismatched miRNA-21, (d)

642

miRNA-141, (e) miRNA-155, (f) miRNA-182, (g) miRNA-143, and (h) the mixture

643

of a–g. The concentration of RNA was 10 pM in all experiments.

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Langmuir

Table 1. Sequence Information for the Nucleic Acids Used in This Study.

Name

Sequences (5′ to 3′)

S1:S2

NH2-(CH2)6-TCAACATCAGTCTGATAAGCTATTCGACATCTAACCTAGCTCACTGAC

miRNA-21

UAGCUUAUCAGACUGAUGUUGA

Single-base mismatched miRNA-21

UAGCGUAUCAGACUGAUGUUGA

Three-base mismatched miRNA-21

UAGCGUAUCCGACUGAUAUUGA

H1

HS-TTTGTCAGTGAGCTAGGTTAGATGTCGCCATGTGTAGACGACATCTAACCTAGCCCTTGT

H2 (C5-5)

AGATGTCGTCTACACATGGCGACATCTAACCTAGCCCATGTGTAGATTTCCCCCTTAATCCCCC

H2 (C6-6)

AGATGTCGTCTACACATGGCGACATCTAACCTAGCCCATGTGTAGATTTCCCCCCTTAATCCCCCC

H2 (C7-7)

AGATGTCGTCTACACATGGCGACATCTAACCTAGCCCATGTGTAGATTTCCCCCCCTTAATCCCCCCC

H2 (C8-8)

AGATGTCGTCTACACATGGCGACATCTAACCTAGCCCATGTGTAGATTTCCCCCCCCTTAATCCCCCCCC

miRNA-141

UAACACUGUCUGGUAAAGAUGG

miRNA-155

UUAAUGCUAAUCGUGAUAGGGGU

miRNA-182

UUUGGCAAUGGUAGAACUCACACU

miRNA-143

UGAGAUGAAGCACUGUAGCUCA

The mutant bases are highlighted in the boxes, and the italic bold letters represent the cluster-nucleation sequences used for Ag NC synthesis.

27


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Scheme 1

Figure 1 28


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Figure 2

29


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3

Figure 4

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Figure 5

Table 2. Comparison of analytical parameters of different methods for miRNA-21 detection. Analytical method

Linear range

Detection limit

Reference

Fluorescent spectroscopy

0.01–2.0 nM

4.2 pM

41

Surface plasmon resonance

10 fM–100 pM

3 fM

42

Chemiluminescence

0.5–500 pM

0.2 pM

43

Colorimetric analysis

10 nM–0.98 µM

3.2 nM

44

Up-converting luminescence

20 fM–4 pM

12 fM

45

Surface-enhanced Raman spectroscopy

1 fM–100 pM

393 aM

46

Electrochemical detection

1.0 pM–10.0 nM

0.26 pM

47

Photoelectrochemical biosensor

5 fM–5 pM

1.67 fM

48

SPR imaging

0.5–200 pM

0.15 pM

49

Polyacrylamide gel electrophoresis

50 pM–8 nM

10 pM

50

Electrochemiluminescence

0.1 fM–100 pM

22 aM

51


0.02–120 pM

6.3 fM

52


1 fM–100 pM

600 aM

This work

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For Table of Contents Only

An efficient platform was explored for the highly sensitive detection of miRNA-21 based on a cytosine-adjusted electrochemiluminescence resonance energy transfer system in combination with a dual signal amplification strategy.

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Page 32 of 32

Construction of a Cytosine-Adjusted Electrochemiluminescence

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