Label-free and amplified electrogenerated chemiluminescence biosensing for the detection of thymine DNA glycosylase activity using DNA-functionalized gold nanoparticles triggered hybridization chain reaction

Accepted Manuscript Label-free and amplified electrogenerated chemiluminescence biosensing for the detection of thymine DNA glycosylase activity using DNA-functionalized gold nanoparticles triggered hybridization chain reaction Wanqiao Bai, Yingying Wei, Yuecheng Zhang, Lin Bao, Yan Li PII:

S0003-2670(19)30138-2

DOI:

https://doi.org/10.1016/j.aca.2019.01.053

Reference:

ACA 236555

To appear in:

Analytica Chimica Acta

Received Date: 17 November 2018 Revised Date:

18 January 2019

Accepted Date: 24 January 2019

Please cite this article as: W. Bai, Y. Wei, Y. Zhang, L. Bao, Y. Li, Label-free and amplified electrogenerated chemiluminescence biosensing for the detection of thymine DNA glycosylase activity using DNA-functionalized gold nanoparticles triggered hybridization chain reaction, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.01.053. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

Label-free

and

amplified

electrogenerated

chemiluminescence

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biosensing for the detection of thymine DNA glycosylase activity

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using DNA-functionalized gold nanoparticles triggered hybridization

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chain reaction

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Wanqiao Baia, 1, Yingying Wei a, b, 1, Yuecheng Zhangc, Lin Baoa, Yan Lia*

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a

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of Education, College of Chemistry and Materials Science, Northwest University,

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Xi’an, Shaanxi 710127, China

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b

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c

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Analytical Technology and Detection, Yan’an University, Yan’an, Shaanxi 716000,

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China

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Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry

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Shaanxi Railway Institute, Weinan, Shaanxi 714000, China

College of Chemistry and Chemical Engineering, Yanan Key Laboratory of

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* Corresponding author. Fax: +86-29-81535026.

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E-mail address: [email protected] (Y. Li).

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1

These authors contributed equally to this work. 1

ACCEPTED MANUSCRIPT Abstract

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Effective detection of thymine DNA glycosylase (TDG) activity is extremely crucial

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and urgent for epigenetic research. Herein, a novel label-free electrogenerated

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chemiluminescence (ECL) biosensing method was developed for the detection of

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TDG activity using DNA-functionalized gold nanoparticles (DNA-AuNPs) triggered

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hybridization chain reaction (HCR). In this assay, the thiol modified hairpin probe

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DNA (hp-DNA) with 5’ overhangs and one mismatched base pair of guanines:

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thymine (G: T) in the stem part was boned onto gold electrode. TDG specifically

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removed T base of the G: T mismatch to produce apyrimidinic (AP) sites through the

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N-glycosidic bond hydrolysis. The AP site was then cleaved by the catalysis of

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Endonuclease IV (EnIV) to generate dsDNA containing a free 3’ end in the long

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sequence, which serves as a complementary sequence to hybridize with the specific

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sequence (ssDNA1) of DNA-AuNPs. Then, the functionalized DNA-AuNPs with

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initiator strands (ssDNA2) could trigger HCR to form nicked double helices DNA

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polymer which can embed numerous ECL indicator, Ru(phen)32+, resulting in

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significantly increased ECL signal. The proposed strategy combined the amplification

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function of DNA-AuNPs triggered HCR and the inherent high sensitivity of the ECL

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technique, a detection limit of 1.1 × 10-5 U/µL (0.0028 ng/mL) for TDG determination

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was obtained. In addition, this method was successfully applied to evaluate TDG

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activity in cancer cell, which provides great possibility for TDG activity assay in

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related clinical diagnostics.

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Keywords: Electrogenerated chemiluminescence; Thymine DNA glycosylase; Tris(1,

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10-phenanthroline) ruthenium; DNA-functionalized AuNPs; Hybridization chain

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reaction

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ACCEPTED MANUSCRIPT 1

1. Introduction Thymine DNA glycosylase (TDG) plays an extremely essential role in defense of

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genetic mutations, maintenance of genetic integrity and study of DNA active

4

demethylation mechanism [1]. TDG can selectively remove the mismatched base to

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generate apyrimidinic (AP) site through N-glycosidic bond hydrolysis and

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subsequently initiates the base replacement by downstream base excision repair (BER)

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pathway both in vitro and in mammalian cells [1-5]. In the BER pathway, TDG can

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remove thymine (T) moieties of guanine : thymine (G : T) mismatched base pairs

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which were formed during process of 5-methylcytosine (5-mC) deamination [6, 7],

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some other lesions can also be removed, such as uracil (U) from G : U mismatch and

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5-hydroxymethyluracil (5-hmU) from G : 5hmU mismatch [8, 9]. In DNA active

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demethylation process, TDG can abscise 5-formylcytosine and 5-carboxylcytosine

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which were formed via sequential oxidation of 5-mC [1,10]. Since TDG has such

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important biological functions, it is crucial to search for and develop a method for

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TDG analysis with high sensitivity, selectivity and convenience.

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Until now, some approaches have been reported to assess TDG activity based on different

principles

or

mechanisms,

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electrophoresis [12-15], fluorescence assay [16-18], electrochemical method [19] etc.

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Although these methods present respective advantages, some limitations still exist.

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Time consumption in gel electrophoresis, short lifetime of fluorescence in organic

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fluorescent compounds, unsatisfying sensitivity for electrochemical method are the

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drawbacks of these methods. It is of great importance to choose the appropriate

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method when detecting a target. Electrogenerated chemiluminescence (ECL) is a

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process in which highly reactive species are applied with high voltage at electrodes

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interface and arousing high-energetic electron transfer reactions which transformed

among

these

methods

including

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ACCEPTED MANUSCRIPT reactive species into excited state that emitting light [20, 21]. Over the past decades,

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biosensors based on ECL have received increasing concern because of their high

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sensitivity, fast detection, easy operation and simple device. To our best knowledge,

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ECL biosensing methods for the detection of TDG have not been reported yet.

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Therefore, the exploration of a novel ultrasensitive ECL biosensing method to

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evaluate TDG activity is highly desirable.

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Always, some extra effort may make great progress in sensing platform, for

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example, Ma’s group discovered luminescent iridium (Ⅲ) complexes which were

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highly selective for G-quadruplex DNA and Al3+, then they utilized these complexes

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to develop a series of G-quadruplex-based probes and chemosensor respectively for

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the detection of disease-related targets [22-25]. Some other transition-metal

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complexes with special properties were subsequently exploited [26-27], combining

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with appropriate techniques or strategies, high-performance sensing platform were

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fabricated for potential therapeutic assessment. For purpose of improving detection

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sensitivity, a series of signal amplification strategies have been applied in the

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construction of DNA sensing platform. Among them, bio-barcode amplification (BCA)

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strategy in sandwich DNA sensing has emerged as an effective way [28-30]. This

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strategy using oligonucleotide modified gold nanoparticles (AuNPs) to hybridize

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target DNA, then target DNA can concatenate magnetic beads through a biotin

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modified DNA probe, sandwiching the targets to accomplish detection. However, the

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conventional BCA-based assay needs to release the barcode DNA strands and

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immobilize it on a microarray, which may lead to cross hybridization and increase the

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experimental complexity [31, 32]. Recently, a new kind of BCA-based assay, termed

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DNA-functionalized gold nanoparticles (DNA-AuNPs), was exploited to achieve the

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sensitivity of polymerase chain reaction (PCR) without enzymes [33, 34]. The

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DNA-AuNPs conjugate was prepared by AuNPs and two-component ssDNA, one of

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the ssDNA components is used to recognize the target, while the other serves as

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bar-code DNA strands to generate signals that reducing the cross-reaction of targets

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with DNA loaded on the same AuNP [35, 36]. Furthermore, hybridization chain

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reaction (HCR) is an isothermal nucleic acid amplification strategy, which has also

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been shown to be as sensitive as PCR [37-39]. The conception of HCR was first

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introduced for the detection of DNA in 2004 [40]. Two kinds of different DNA hairpin

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probes which contain complementary fragments each other can maintain stable forms

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in the hybridization solution. Nevertheless, when initiator DNA strands are added, a

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succession of hybridization events will be triggered because the two partially

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complementary DNA hairpin probes hybridize with each other to form DNA polymers

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with nicked double helices. Since each counterpart of the DNA initiators can touch off

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a HCR process, leading to combine a large amount of oligonucleotides, which

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supports enormous possibilities for signal amplification. Moreover, HCR is a unique

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assembly process which can work under common conditions without enzyme or

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special equipment. These salient properties make HCR an absorbing strategy in

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construction of sensing platform for detection of DNA [41, 42], proteins [37], cells

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[43] and metal ions [44].

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Herein, a label-free and highly sensitive ECL biosensing method for TDG

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activity detection was proposed based on signal amplification strategy of

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DNA-AuNPs triggered HCR with Ru(phen)32+ as ECL indicator. In this paper, AuNPs

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were functionalized with two kinds of different DNA sequences (denoted as ssDNA1

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and ssDNA2) to form DNA-AuNPs structure. Sequences ssDNA1 in the structure can

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hybridize with hairpin probe DNA (hp-DNA) which was incubated with TDG and

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Endonuclease IV (EnIV) on the electrode surface, leading to capture of DNA-AuNPs

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ACCEPTED MANUSCRIPT onto the electrode. Whereas ssDNA2 can act as initiator strands to trigger HCR, the

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secondary amplification element, forming a nicked double helices DNA polymer.

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Consequently, numerous Ru(phen)32+ molecule, an ECL signal reporter, which can

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readily insert into the groove of nicked double helices DNA polymer to generate

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amplified ECL signal [45, 46]. Coupling DNA-AuNPs with HCR amplification

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strategy, we developed the ECL biosensing method which could be applied to detect

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the TDG activity with high sensitivity, reliability and robustness.

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2. Experimental

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2.1. Reagents and materials

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Chloroauric acid (HAuCl4), tris(2-carboxyethyl) phosphinehydrochloride (TCEP,

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98%), dichlorotris (1, 10-phenanthroline) ruthenium (II) hydrate ([Ru(phen)3]2+),

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tripropylamine (TPA), 6-mercapto-1-hexanol (MCH), methyl methanesulfonate

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(MMS, 99%) were bought from Sigma-Aldrich (U.S.A.). Other common reagents

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were from Sinopharm Chemical Reagent Co., Ltd. (China). TDG and reaction buffer

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were obtained from R&D system (U.S.A.). EnIV, uracil-DNA glycosylase (UDG),

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8-oxoguanine DNA glycpsylase (hOGG1), bovine serum albumin (BSA) and low

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range DNA marker were acquired from New England Biolabs Ltd. (U.S.A.). Human

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immunoglobulin G (IgG) and prostate specific antigen (PSA) was from Beijing

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Biosynthesis Biotechnology Ltd. (China) and Fitzgerald Industries International, Inc.

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(U.S.A.), respectively. All chemicals utilized were of analytical grade. Ultrapure water

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(18.2 MΩ cm) was supplied throughout the experiment. All oligonucletides were

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synthesized by Shanghai Sangon Biotechnology Co. Ltd. (China). Two DNA hairpin

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probes (H1, H2) were adopted from Chen’s work [38]. The sequence of hairpin probe

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DNA (hp-DNA), ssDNA1, ssDNA2, H1, H2, and the control probe DNA (cp-DNA)

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were listed in Table S1 in Supporting Information. Prior to use, each hairpin DNA was

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ACCEPTED MANUSCRIPT heat-treated at 90 °C for 5 min and cooled naturally later on. The pH of

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phosphate-buffered saline (PBS) used in the detection was 7.4 unless specifically

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stated. 0.1 M PBS (Buffer I) was used as immobilization buffer. 0.5 M NaCl was

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added into 0.1 M PBS to prepare HCR buffer (Buffer II). The solution for

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electrochemical impedance spectroscopy (EIS) test was 0.1 M PBS with 5 mM

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[Fe(CN)6]3−/4−. The ECL detection solution was 0.1 M PBS including 50 mM TPA.

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2.2 Apparatus

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The ECL measurements were monitored by an MPI-E type ECL analyzer (Xi’an

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Remex Electronics, China). EIS was taken with a CHI 660D electrochemistry workstation

Instruments,

China).

Both

electrochemical

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measurements and ECL were carried out with traditional three-electrode system:

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Ag/AgCl (saturated KCl), Pt electrode and Au electrode (Φ = 2 mm) worked as

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reference, counter and working electrode, respectively. A Tecnai G2 F20 microscope

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(FEI Co., U.S.A.) was used to supply transmission electron microscopy (TEM). The

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dynamic light scattering (DLS) data was obtained by Zetasizer Nano ZS (Malvern

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Instruments, Southborough, U.K.). The UV-vis absorption spectra were carried out

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utilizing a Shimadzu UV-2550 spectrometer (Japan). The polyacrylamide gel

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electrophoresis (PAGE) was carried out with a Bio-rad slab electrophoresis system

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(Bio-Rad, U.S.A.).

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2.3. Preparation of DNA-AuNPs

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It has been reported that AuNPs with a diameter of ~13 nm possess interesting

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physical and chemical properties, can be densely functionalized with oligonucleotides

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[47, 48]. So AuNPs of this size were used in the preparation of DNA-AuNPs. Firstly,

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100 mL of chloroauric acid (0.01 wt %) aqueous solution was added into a clean flask

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and heated to boil, and then 4 mL of sodium citrate solution (1 wt %) was fast dripped

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ACCEPTED MANUSCRIPT 1

into the flask under stirring. When the colorless solution gradually changed to wine

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red, the boiling state was maintained for another 30 min. The obtained solution

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containing AuNPs were cooled down to room temperature for further use [36, 49]. DNA-AuNPs were prepared according to published literature [35, 50]. The glass

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vials were cleaned thoroughly before use. Then, 3.5 µL of 1 mM ssDNA2 and 5 µL of

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0.1 mM ssDNA1 were mixed and activated with 4 µL TCEP (100 mM) and 1 µL

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acetate buffer (500 mM, pH 5.2) for 1 hour [32]. Next, 2 mL of freshly prepared

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AuNPs solution was transferred to the above mixture and shaken slightly in dark

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overnight at room temperature. Afterward, 20 µL Tris-acetate buffer (500 mM, pH 8.2)

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and 200 µL NaCl (1 M) was dropwise dripped into the glass vial, the obtained mixture

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was incubated for another 24 h in dark. Eventually, the mixture was centrifuged for 20

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min to eliminate excess DNA. The precipitate was washed and centrifugated for three

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times. The resulting DNA-AuNPs were redispersed into 2 mL of Buffer II and

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reserved it at 4 °C in dark for later detection.

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2.4. Fabrication of ECL biosensing electrode

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The Au electrode was cleaned to a mirror by successively polishing with 0.3 and

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0.05 µm Al2O3 and wishing with water. Afterwards, the electrode was

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electrochemically cleaned using H2SO4 solution (0.1 M) with a linearly scanning

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potential (0.2 ~1.6 V) to obtain stable cyclic voltammograms. Subsequently, the

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electrode was incubated with 10 µL hp-DNA (1 µM) in Buffer I for overnight at room

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temperature. Then, the surface of electrode was rinsed thoroughly with PBS to

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remove the unbound hp-DNA. Finally, the possible remaining active sites on the

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modified electrode were blocked with MCH for 1 hour to obtain MCH/hp-DNA/Au

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electrode.

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2.5. ECL measurements

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ACCEPTED MANUSCRIPT ECL biosensing electrodes were incubated by 10 µL TDG solutions with various

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concentrations for 100 min at 65 °C to identify the mismatched G: T base pair and

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remove T. Then, the resulting TDG/MCH/hp-DNA/Au electrode was incubated with

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10 µL 0.33 U/µL EnIV for 2 h at 25 °C to generate the double-stranded DNA (dsDNA)

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with a free 3’ end in the long sequence, rinsing the modified electrode with pure water.

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Subsequently, 10 µL DNA-AuNPs dispersion were modified onto the above electrode

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surface for 90 min. After rinsing to eliminate unbounded DNA-AuNPs with PBS (pH

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7.4), the modified electrodes were hatched with 10 µL Buffer II containing 1 µM (H1

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+ H2) mixture for 2 hours at room temperature and then rinsed with PBS to wipe off

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residual free DNA. Ultimately, 10 µL Ru(phen)32+ solution (2 mM) diluted with PBS

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was dropped onto (H1 + H2)/DNA-AuNPs/EnIV/TDG/MCH/hp-DNA/Au electrode

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for 5 hours. After thoroughly rinsing with Buffer II, the electrode was readied for ECL

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measurements. The test solution was 1 mL PBS (0.1 M) with 50 mM TPA as

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coreactant. The ECL measurement was conducted over a scanning range of 0 ~ 1.25 V,

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the voltage of photomultiplier tube (PMT) was set at 900 V in the detection process.

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2.6. Gel electrophoresis

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12% native PAGE was performed to verify the interactions between different

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DNA and ensure cascade signal amplification. In gel electrophoresis assay, 10 µL of

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the various DNA samples were mixed with 2 µL 6 × DNA loading buffer and kept for

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3 min so that the dye can completely integrate with DNA samples. The PAGE was

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conducted in the slab electrophoresis system with 0.5 × Tris-borate-EDTA buffer as

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electrophoresis buffer at room temperature, the voltage was constant 110 V. The result

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was visualized under UV light and photographed with gel image system.

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3. Results and discussions

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3.1. Principle of the strategy

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ACCEPTED MANUSCRIPT The detection scheme of the proposed method is illustrated in Scheme 1. We

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divided the detection procedure into three phases: (Ⅲ) Converting hp-DNA into

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ds-DNA. In this phrase, the thiol modified hp-DNA with 5’ overhangs and single G: T

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mismatch in the stem part was immobilized on Au electrode via Au-S bond. When

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TDG was added, T base of the G: T mismatch could be specifically removed to

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produce AP sites through the N-glycosidic bond hydrolysis. Then, EnIV was

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introduced to cleave intact AP sites through the hydrolysis of the 5’ phosphodiester

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bond of the AP site, leading to the hp-DNA converted into dsDNA with a free 3’ end.

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(Ⅲ) Trigger the HCR process. In this phrase, DNA-AuNPs functionalized with

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ssDNA1 and ssDNA2 were introduced, the sequence of ssDNA1 was specifically

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hybridized with the long sequence of dsDNA, resulting capture of DNA-AuNPs on

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the electrode. At the same time, two kinds of hairpin probes H1 and H2 both possess

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12 base pairs stem and a hexanucleotide loop were adopted, with a hexanucleotide

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sticky end which is complementary to the loop of each other. ssDNA2 on

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DNA-AuNPs act as bar-code strand, could hybridize with H1 at the sticky end

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accompanied with a strand displacement interaction, which opened H1 loop and

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exposed the rest sequence of H1. The newly exposed fragment will bind with sticky

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end of H2 and then untie H2 loop. The exposed part of H2 was identical in sequence

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to ssDNA2, which will sequentially hybridize with the complementary segment in H1.

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In this way, each bar-code strand propagates a HCR event between alternating H1 and

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H2, resulting in formation of nicked double helices DNA polymers. (Ⅲ) ECL

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performance. In this phase, numerous ECL signal indicators, Ru(phen)32+ molecules,

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could be readily embedded into the grooves of double helices DNA polymers, which

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generated amplified ECL signals. The ECL signal intensity is directly proportional to

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the quantity of TDG so it serves as the quantitative parameter for TDG activity

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ACCEPTED MANUSCRIPT detection. With the advantage of DNA-AuNPs triggered HCR signal-amplification

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strategy, an ECL biosensing platform for TDG activity determination with

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ultra-sensitivity was proposed and applied in cancer cells detection.

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Scheme 1. Schematic depiction of (A) preparation of DNA-AuNPs and (B) the fabrication process

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of the ECL biosensing platform for the detection of TDG activity based on DNA-AuNPs triggered

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HCR.

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3.2. Characterization of AuNPs and AuNPs complexes The modification of AuNPs was investigated by UV-vis absorption spectroscopy

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(in Figure 1). For AuNPs, a characteristic absorption peak (520 nm) which accords

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with the typical surface plasmon resonance band of 13~14 nm AuNPs [51] was

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observed (curve a). While the UV-vis spectrum of DNA-AuNPs shows two absorption

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peaks at 525 and 260 nm (curve b). This slightly red-shift of AuNPs may ascribe to

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the fact that a slight decrease of the average distance between gold particles caused by

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a modest inter-molecular dimerization of thiol DNA, moreover, the centrifugation

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during the preparation of DNA-AuNPs may also affect the particle size distribution 11

ACCEPTED MANUSCRIPT [52]. The presence of absorption peak at 260 nm corresponding to the typical DNA

2

absorption peak suggests the successful modification of DNA on AuNPs surface.

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When DNA-AuNPs were mixed with H1 and H2, the absorption peak around 260 nm

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was strongly increased (curve c) comparing with that of DNA-AuNPs, indicating the

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successful initiation of HCR on the surface of AuNPs. Meanwhile, the coverage of

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DNA on AuNPs was estimated to be 65 DNA strands per AuNP according to a

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UV-visible-based method reported in previous research [53].

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Figure 1. UV-vis spectra of (a) AuNPs, (b) DNA-AuNPs and (c) DNA-AuNPs after treatment

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with 1 µM H1 and 1 µM H2 for 1 h.

The morphology and dispersity of AuNPs without and with DNA modification

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were shown in Figure 2a-c by TEM images. The prepared AuNPs and DNA-AuNPs

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were monodispersed spherical nanoparticles with a narrow particle size distribution.

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The remarkable monodispersity of DNA-AuNPs is a prerequisite to achieve

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controllable and precise DNA sensing signal amplification [54]. Furthermore, the 12

ACCEPTED MANUSCRIPT average hydrodynamic sizes of AuNPs, DNA-AuNPs and DNA-AuNPs mixed with

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(H1 + H2) were determined by DLS as presented in Figure 2d-f, the hydrodynamic

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size of them are about 13.54, 24.36 and 68.06 nm, respectively. The results confirmed

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that the increasing in size of AuNPs is caused by DNA assembly, a similar result has

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been reported by a previous research [55].

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Figure 2. TEM images and DLS results of AuNPs (a and d), DNA-AuNPs (b and e), DNA-AuNPs

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(c and f) after treatment with 1 µM H1 and 1 µM H2 for 1 h.

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3.3. Electrochemical characterization

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EIS was used to monitor the stepwise fabrication process of modified electrode

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with [Fe(CN)6]3−/4− as redox probe. As displayed in Figure S1 (in Supporting

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Information), the bare Au electrode shows a near-straight line of EIS (curve a, 19 Ω),

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indicating the excellent conductivity of bare Au electrode. After hp-DNA was

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assembled on the Au electrode, a larger electron transfer resistance (Ret) can be

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observed (curve b, 331 Ω), which may be caused by the electrostatic repulsion

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interaction between [Fe(CN)6]3−/4− probe and phosphate backbone of DNA strands

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with the same electronegativity. Ret value further increased after MCH blocking the

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ACCEPTED MANUSCRIPT remaining active sites (curve c, 701 Ω). When the modified electrode was

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subsequently reacted with TDG (curve d, 692 Ω) and EnIV (curve e, 417 Ω), Ret

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greatly decreased in turn. It suggests that the decrease in Ret is attributed to the

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removal of T base and the formation of nick under the function of TDG and EnIV,

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which successfully transformed hp-DNA into dsDNA. With further incubation with

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DNA-AuNPs, Ret increased (curve f, 824 Ω) again, which could be ascribed to the

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numerous negatively charged DNA strands on AuNPs which kept [Fe(CN)6]3−/4−

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probes away from the contact interface and also steric hindrance effect. Finally,

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DNA-AuNPs triggered HCR introducing more negatively charged DNA strands to the

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electrode interface, thus a larger Ret was obtained (curve g, 2011 Ω). These results

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verified the successful fabrication of the biosensing electrode.

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3.4. Feasibility study

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In order to validate the feasibility of the ECL biosensing platform, two native

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PAGEs were conducted. As shown in Figure 3A, hp-DNA exhibits two separate bands

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(lane 1), which may due to the aggregates formed by disulfide bonds between DNA

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probes themselves. After treating hp-DNA with 1 × 10-2 U/µL TDG, the two bands

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shift slightly (lane 2), indicating the DNA structure has undergone minor changes by

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the base excise process of TDG. When 0.33 U/µL EnIV was further added, a broader

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band came out (lane 3) compared with that of lane 2, which may be caused by

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changes in DNA structure. We deduce that after the cleavage of EnIV, the hairpin

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probe DNA had transformed into a double-stranded hybridization structure [16].

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These results indicate that TDG and EnIV can specifically recognize and cleave

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hairpin probes containing a G: T mismatch in the stem part, leading to the formation

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of DNA double helix structures. As another PAGE shown in Figure 3B, lane 1 and

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lane 2 are electrophoretic bands of H1 and H2, respectively. Although H1 and H2

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ACCEPTED MANUSCRIPT contained partly complementary sequences for each other, the emission band of their

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mixture become broader (lane 3), demonstrating H1 and H2 were hybridized together

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only in a small scale. A great amount of unreacted H1 and H2 monomer still existed at

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the bottom of lane 3, and the emission band is brighter. When initiator strands of

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ssDNA2 were further added, largely diffused bands can be observed easily,

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meanwhile, the bands corresponding to H1 and H2 almost disappeared (lane 4),

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manifesting ssDNA2 initiated the HCR process successfully. When DNA-AuNPs was

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added instead of ssDNA2 to blend with (H1 + H2), an emission band of DNA

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polymer with high molecular weight is displayed at the top of lane 5, implying the

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successful initiation of HCR by ssDNA2 on AuNPs surface. The above results

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confirm that HCR was successfully triggered by DNA-AuNPs.

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Figure 3. (A) Results of PAGE: lane M, DNA marker; lane 1, 1 µM hp-DNA; lane 2, 1 µM

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hp-DNA + 1 × 10-2 U/µL TDG; lane 3, 1 µM hp-DNA + 1 × 10-2 U/µL TDG + 0.33 U/µL EnIV. (B)

15

PAGE demonstration of DNA-AuNPs triggered HCR: lane M, DNA marker; lane 1, 1 µM H1;

16

lane 2, 1 µM H2; lane 3, mixture of 1 µM H1 and 1 µM H2; lane 4, the presence of 1 µM ssDNA2

17

with a mixture of 1 µM H1 and 1 µM H2; lane 5, the presence of DNA-AuNPs with a mixture of 1

15

ACCEPTED MANUSCRIPT 1

µM H1 and 1 µM H2 (all the samples were mixed and incubated at room temperature for 1 hour).

2

The corresponding ECL signals of the diversely modified electrode were also

4

recorded. As shown in Figure 4, when hp-DNA, EnIV, DNA-AuNPs, H1 and H2 were

5

present but without the target TDG, a weak ECL signal (curve a, 1000 a.u.) is

6

observed. This is because hp-DNA cannot be cleaved by EnIV to form dsDNA

7

without AP sites, and DNA-AuNPs can be ineffectively captured on the electrode by

8

hybridization. As a result, the weak interaction of Ru(phen)32+ generated a relatively

9

low ECL background signal. When EnIV was absence but TDG existed, a little higher

10

ECL signal (curve b, 1483 a.u.) can be observed, which may due to the weak lyase

11

activity of TDG to cleave hp-DNA [16], producing little dsDNA to complete the

12

follow-up process. In addition, the dual signal amplification capability of the protocol

13

was also studied. After incubating Au electrode with hp-DNA, TDG, EnIV and the

14

mixture of (H1 + H2) (curve c, 1203 a.u.) or DNA-AuNPs (curve d, 1879 a.u.), the

15

ECL signal were improved compared with curve a, but still weaker than that of the

16

both of them involved system (curve e, 5366 a.u.). The above results indicate the dual

17

amplification effect of this proposed method is excellently efficient and our design is

18

reasonable.

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1 2

Figure

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H2/DNA-AuNPs/EnIV/MCH/hp-DNA/Au

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TDG/MCH/hp-DNA/Au electrode, (c) (H1 + H2)/EnIV/TDG/MCH/hp-DNA/Au electrode (d)

5

DNA-AuNPs/EnIV/TDG/MCH/hp-DNA/Au

6

H2)/DNA-AuNPs/EnIV/TDG/MCH/hp-DNA/Au electrode. The ECL measurements were

7

performed in 0.1 M PBS with 50 mM TPrA. Potential scan from 0.0 to +1.25 V at the scan rate of

8

50 mV/s. The concentration of TDG, H1 and H2 were 5 × 10-4 U/µL, 1 µM and 1 µM,

9

respectively.

11 12

measurements

for

different

electrode,

modified (b)

electrode,

(H1

electrodes: +

(e)

(a)

H1

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H2)/DNA-AuNPs/

(H1

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3.5. Optimization of the reaction conditions The reaction temperature of TDG has a crucial effect on ECL signal, which was

13

optimized with a constant concentration of 5 × 10-5 U/µL (Figure S2A). The result

14

displays that ECL intensities increase gradually along with the increase of reaction

15

temperature from 25 to 65 Ⅲ, however, further increasing reaction temperature to

16

75 Ⅲ exhibited a little decreased ECL intensity. This can be attributed to the fact that

17

ACCEPTED MANUSCRIPT high temperature is not conducive to TDG activity. Therefore, 65 Ⅲ was chosen as the

2

optimized reaction temperature for TDG. In addition, the reaction time of TDG is also

3

a considerable influencing factor. The dependence of ECL intensities on reaction time

4

of TDG was also studied (Figure S2B). A significant increase of ECL intensity was

5

found from 30 to 100 min, while no obvious ECL intensity enhancement was gained

6

for longer time. This result shows a saturated reaction between the TDG and the

7

hp-DNA on the biosensing electrode surface. Therefore, 100 min reaction time was

8

decided in the following experiments.

9

3.6. Detection of TDG activity

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Under the optimum condition, the proposed method for the detection of TDG

11

activity was evaluated. As can be seen from Figure 5, ECL intensities logarithmically

12

correlated with the increasing TDG concentrations in the range from 5 × 10-5 U/µL to

13

1 × 10-2 U/µL (Figure 5B). The regression equation between ECL intensities and TDG

14

concentrations is IECL (a.u.) = 2594.9 lgC (U/µL) + 13703 with a correlation

15

coefficient of 0.9986. At a signal-to-noise ratio of 3, the detection limit (LOD) of this

16

ECL biosensing method is calculated to be 1.1 × 10-5 U/µL (0.0028 ng/mL). In

17

comparison with other reported detection methods of TDG activity, the proposed ECL

18

biosensing method exhibits a lower LOD and broader detection concentration range

19

(Table S2). The excellent analytical performance can be attributed to the amplification

20

strategy of DNA-AuNPs triggered HCR, which endows higher double helices DNA

21

polymers capacity on the electrode surface and enable them to intercalate numerous

22

Ru(phen)32+ into the grooves.

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Figure 5. (A) ECL intensity obtained with different concentration of TDG (a-i): 0 U/µL, 5 × 10-5

4

U/µL, 8 × 10-5 U/µL, 2 × 10-4 U/µL, 3 × 10-4 U/µL, 5 × 10-4 U/µL, 1 × 10-3 U/µL, 3 × 10-3 U/µL, 1 ×

5

10-2 U/µL; (B) Plot of ECL intensity versus TDG concentration. Insert: the corresponding

6

calibration curve.

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3.7. Reproducibility, specificity and stability

To verify the reproducibility of this method, intra- and inter-assays (n = 5) were

10

conducted with 5 × 10-4 U/µL TDG as a model, and the relative standard deviation

11

(RSD) was used to assess the reproducibility. The intra-assay RSD was evaluated by

12

detecting target TDG through five replicate measurements under the same

13

experimental conditions while the inter-assay RSD was estimated by detecting target

14

TDG using five designed biosensing electrodes fabricated in different batches under

15

the same conditions. The obtain RSDs of intra- and inter-assay were 3.3 % and 5.4 %,

16

respectively. The results suggest the proposed ECL biosensing method exhibited an

17

acceptable reproducibility for TDG detection. Moreover, specificity is an important

18

factor to evaluate the feasibility of the proposed biosensing platform in real samples

19

[51]. Two base-specific glycosylases including hOGG1 and UDG, three proteins

20

including IgG, PSA, BSA as well as cp-DNA containing perfectly matched G: C pair

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ACCEPTED MANUSCRIPT were utilized as the possible interferences to confirm the specificity of this method

2

(Figure S3A). The result shows that no significant ECL response can be observed

3

except that for TDG, demonstrating that the proposed method has an excellent

4

specificity for TDG activity detection and a promising application capability in real

5

samples. Furthermore, the stability of the proposed strategy was also studied by

6

recording the ECL intensity after storage at 4 °C for different time intervals in the unit

7

of day(s). As presented in Figure S3B, the ECL intensity on the electrode decreases by

8

14.5% after fifteen days compared to the ECL intensity without time delay under the

9

same concentration of TDG. Figure S3C displays the ECL-time curve incubated with

10

TDG (5 × 10-4 U/µL) under consecutive cyclic potential scans for 15 cycles with an

11

RSD of 1.8 %, which further demonstrates that the prepared ECL platform has good

12

stability in the optimizing experimental condition.

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3.8. Detection of TDG activity in cancer cells

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Considering the practicability, TDG activity in human breast cancer MCF-7 cells

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lysates (see more in Supporting Information) was detected by this proposed

16

biosensing platform. As displayed in Figure 6A, when a lysis buffer was introduced

17

into the ECL biosensing platform instead of TDG, a low background ECL signal can

18

be acquired (strip a). In contrast, the gradually increased ECL intensities were

19

obtained with the increment of MCF-7 cells amount (strip b-f). This result is in

20

accordance with the fact that, with increasing cell number, more active TDG can act

21

on the hp-DNA and generate more AP sites. Thus, after incubation with EnIV,

22

DNA-AuNPs, and mixture of (H1+H2) respectively, a strong ECL signal was

23

obtained. The results confirm that this proposed method has a great potential for the

24

detection of TDG activity in clinical diagnosis. In addition, methyl methanesulfonate

25

(MMS), an alkylating agent, was used as a model inhibitor and introduced into the

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ACCEPTED MANUSCRIPT MCF-7 cells (104) lysates to demonstrate the potential application of this method in

2

screening the inhibitor of TDG, and the result is shown in Figure 6B. As can be

3

observed, as the inhibitor concentration of MMS increased, the corresponding ECL

4

intensity decreased. When the trace MMS as low as 0.01 % was introduced into the

5

MCF-7 cells lysates, the ECL intensity decreased by about 80 %, which means the

6

activity of TDG was greatly inhibited. The result confirms that the proposed method

7

could be promising to evaluate the inhibition ability of inhibitors.

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Figure 6. (A) The ECL intensity obtained from cell extracts with different number of MCF-7 cells:

10

(a) lysis buffer, (b) 10 cells, (c) 102 cells, (d) 103 cells, (e) 5 × 103 cells and (f) 104 cells; (B) The

11

effect of inhibitor on TDG activity.

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4. Conclusions

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In this work, a novel label-free ECL biosensing method was developed to detect

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TDG activity using signal amplification strategy of HCR which was triggered by

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DNA functionalized AuNPs. The label-free ECL biosensing platform has been

17

constructed for TDG activity detection for the first time by utilizing of Ru(phen)32+ as

18

an intercalated signal indicator. The high sensitivity of ECL method combined with

19

the remarkably amplified effect of the DNA-functionalized gold nanoparticles

20

triggered hybridization chain reaction endows this biosensing method with ultra-high

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ACCEPTED MANUSCRIPT 1

sensitivity and a low detection limit. Additionally, the feasibility of this strategy for

2

TDG detection in MCF-7 cells lysates was also confirmed. This method creates a new

3

horizon for quantitative detection of TDG, and it shows great potential for TDG

4

activity assay in clinical diagnostics and related research.

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Acknowledgements

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Financial support from the National Natural Science Funds of China (Nos. 21675124,

8

21375102 and 201706214), the Natural Science Basic Research Plan in Shaanxi

9

Province of China (No. 2016JM2021) and the Excellent Doctoral Dissertations Project of Northwest University (No. YYB17013) are acknowledged.

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version.

14

Cell culture and preparation of cell extracts; The sequence of the synthesized

15

oligonucleotides (Table S1); EIS of different modified electrodes (Figure S1);

16

Optimization of experimental conditions (Figure S2); Selectivity and stability

17

investigation (Figure S3); Analytical performance compared with other works (Table

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S2) and Procedure of TDG activity assay, as noted in the text.

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[55] W.J. Wang, J.J. Li, K. Rui, P.P. Gai, J.R. Zhang, J.J. Zhu, Sensitive

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electrochemical detection of telomerase activity using spherical nucleic acids

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gold nanoparticles triggered mimic-hybridization chain reaction enzyme-free dual

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signal amplification, Anal. Chem. 87 (2015) 3019-3026.

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Highlights


A label-free ECL biosensing method for ultrasensitive thymine DNA glycosylase was developed.




The ECL biosensing method is based on the dual-amplification of DNA-AuNPs

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and HCR.

A detection limit of 1.1 × 10-5 U/µL was obtained for thymine DNA glycosylase.




The method was applied to evaluate TDG activity in cancer cell.

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Declaration of Interest Statement The authors declared that they have no conflicts of interest to this work.

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We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work

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submitted