Affinity-Mediated Homogeneous Electrochemical Aptasensor on a

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Affinity-Mediated Homogeneous Electrochemical Aptasensor on Graphene Platform for Ultrasensitive Biomolecule Detection via Exonuclease-Assisted Target-Analog Recycling Amplification Lei Ge, Wenxiao Wang, Ximei Sun, Ting Hou, and Feng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03844 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on January 27, 2016

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Analytical Chemistry

1

Affinity-Mediated Homogeneous Electrochemical Aptasensor on

2

Graphene Platform for Ultrasensitive Biomolecule Detection via

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Exonuclease-Assisted Target-Analog Recycling Amplification

4 5 6 7

Lei Ge, Wenxiao Wang, Ximei Sun, Ting Hou, and Feng Li*

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College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University,

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Qingdao, 266109, People’s Republic of China

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*Corresponding author: Feng Li

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E-mail: [email protected]

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Telephone: +86-532-86080855 1

ACS Paragon Plus Environment


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ABSTRACT: As is well-known, graphene shows remarkable difference in affinity

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toward non-structured single-stranded (ss) DNA and double-stranded (ds) DNA. This

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property makes it popular to prepare DNA-based optical sensors. In this work, taking

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this unique property of graphene in combination with the sensitive electrochemical

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transducer, we report a novel affinity-mediated homogeneous electrochemical

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aptasensor using graphene modified glassy carbon electrode (GCE) as the sensing

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platform. In this approach, the specific aptamer-target recognition is converted into

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ultrasensitive electrochemical signal output with the aid of a novel T7 exonuclease

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(T7Exo)-assisted target-analog recycling amplification strategy, in which the

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ingeniously designed methylene blue (MB)-labeled hairpin DNA reporters are

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digested in the presence of target and, then, converted to numerous MB-labeled long

34

ssDNAs. The distinct difference in differential pulse voltammetry response between

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the designed hairpin reporters and the generated long ssDNAs on the graphene/GCE

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allows ultrasensitive detection of target biomolecule. Herein, the design and working

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principle of this homogeneous electrochemical aptasensor were elucidated, and the

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working conditions were optimized. The gel electrophoresis results further

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demonstrate that the designed T7Exo-assisted target-analog recycling amplification

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strategy can work well. This electrochemical aptasensor realizes the detection of

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biomolecule in a homogeneous solution without immobilization of any bioprobe on

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electrode surface. Moreover, this versatile homogeneous electrochemical sensing

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system was used for the determination of biomolecule in real serum samples with

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satisfying results. 2


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INTRODUCTION

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The sensitive and simple detection of biomolecules has become a major and

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imperative research focus in science and technology for many decades, given its

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myriad applications in environmental monitoring, molecular diagnostics, food and

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agricultural industries, and antibioterrorism etc..1,2 Over the past decade, various

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aptamer-based

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electro-chemiluminescent

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photoelectrochemical aptasensors,10,11 have been developed as an alternative to the

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conventional immunology-based methods for various biomolecules detection. In this

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regard, electrochemical strategy holds great promise as powerful bioanalytical

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technique for biomolecules detection and exhibits several noticeable advantages,12-15

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such as simple instrumentation, rapid response, low cost, high sensitivity, and ease of

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integration into miniaturized devices for scale-up and multiplexed biomolecules

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detection,16-20 which are desirable for point-of care and make them potentially great in

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diseases diagnosis/management, and for conducting fundamental biological studies.

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Up to now, a large majority of modern electrochemical assays rely on solid-phase

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bio-recognition probe immobilization strategies, which play significant roles in the

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assay performances of these electrochemical biosensors, and an inappropriate

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immobilization strategy to fix the bio-recognition probes on electrode surface may

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even result in no signal response.21,22 However, due to the lack of a uniform strategy

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for various bio-recognition probe immobilizations, it would be tedious and difficult to

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immobilize different bio-recognition probes on different electrodes, or even

biosensors,

such

as

aptasensors,5,6

chemiluminescent fluorescent

3


aptasensors,3,4

aptasensors,7-9

and


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simultaneously on the same electrode, limiting the routine use of electrochemical

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biosensors, especially, in multiplexed assay. Furthermore, it has been reported that the

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some nucleases exhibit inhibited catalytic activity towards the immobilized nucleic

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acid substrates, as compared to the free nucleic acid substrates in homogeneous

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solution.23,24 As such, an electrochemical detection strategy that can directly detect

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target biomolecules in homogeneous solution without immobilization of any bioprobe

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would greatly facilitate the development of rapid, cost-effective, reliable, and

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easy-to-use biomolecule assays.

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Recently, varieties of homogeneous DNA-based electrochemical strategies have

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been developed through detecting the diffusion current of quantificationally

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activated/generated methylene blue,25-27 ferrocene,28,29 or other electroactive

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substances30,31 in the homogeneous reactions. However, compared to traditional

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heterogeneous electrochemical biosensors, the aforementioned diffusion-mediated

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homogeneous

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activated/generated electroactive labels, which disperse freely in the whole

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homogeneous solution, to produce current signal, limiting the signal enhancement and

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thus the sensitivity of the assay. It has been reported that graphene shows much higher

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affinity toward non-structured single-stranded DNA (ssDNA) due to the strong π−π

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stacking interaction between the hexagonal cells of graphene and the ring structures in

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nucleobases of ssDNA, while its interaction with double-stranded (ds) DNA, which

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effectively shields its nucleobases in the helical structure, is disfavored.32-34 This

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excellent ssDNA/dsDNA discrimination ability of graphene inspired us to develop a

electrochemical

strategies

can

not

4


efficiently

utilize

the

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novel versatile affinity-mediated homogeneous electrochemical aptasensor to

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overcome the aforementioned problem.

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Graphene has been widely studied as a promising type of electrode material in

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the fields of chemo-/bio-sensing,35-39 because of its high electrical conductivity and

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large surface area. Thus, toward the construction of affinity-mediated homogeneous

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electrochemical aptasensor in this work, graphene is modified on the surface of glassy

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carbon electrode (GCE) and the resultant graphene modified GCE is employed as the

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electrode substrate, which is fabricated through the attachment of graphene sheets on

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the

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homogeneous fluorescent DNA biosensors,40-42 most of the activated/generated

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electroactive substance (methylene blue in this work, MB) labeled ssDNAs could be

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firmly adsorbed on the graphene modified GCE through the strong π−π stacking

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interaction and thus generate high electrochemical signal, while quite low

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electrochemical response is observed when the MB-labeled ssDNAs (MB-ssDNAs)

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hybridize with their complementary ssDNAs to form double helixes, not only

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providing a high signal-to-background ratio, but also increasing the utilization

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efficiency of the quantificationally activated or generated electroactive substances in

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homogeneous solution.

p-aminophenyl-grafted

GCE.

Similar

to

conventional

graphene-based

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In most traditional aptasensors, as is well-known, one or two target molecules

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could only trigger a single target-aptamer binding event, which limits the total signal

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gain and thus shows poor sensitivity. Thus, signal amplification strategy has been

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commonly involved in the design and construction of aptasensors, which could 5



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greatly improve the detection sensitivity.43-46 Nuclease-assisted signal amplification

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strategies, especially the exonuclease-catalyzed degradation reaction of probe DNA,

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have become attractive in the fabrication of aptasensors, which allow one target to

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interact with multiple DNA probes and contribute to ultrahigh detection

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sensitivity.47-53 In this contribution, we designed a novel single-step T7 exonuclease

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(T7Exo)-assisted target-analog recycling circuit with isothermal signal amplification

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ability to generate numerous MB-ssDNAs as the signal output for this

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affinity-mediated homogeneous electrochemical aptasensor.

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In this strategy, a hairpin DNA probe (HP1, containing the aptamer sequence)

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and a MB-labeled hairpin DNA reporter (HP2) were ingeniously designed, both of

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which consist of a protruding ssDNA fragment at their 5’-end to prevent themselves

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from being digested by T7Exo, which specifically cleaves duplex DNA from blunt or

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recessed 5’ termini,54 in the absence of the target biomolecule. The recognition of

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aptamer sequence in HP1 to target biomolecule switches on the hybridization between

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the formed HP1@target complex and HP2 and, then, initiates the digestion of HP2 in

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the presence of T7Exo, which not only generates a long MB-ssDNA, but also releases

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the HP1@target complex. Both of the released HP1@target complex and the

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generated MB-ssDNA possess the same complementary sequence to the 5’-protruding

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ssDNA fragment of HP2, and thus could bind to other HP2s to initiate next

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digestion-releasing cycles, which leads to recycling amplification

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electrochemical signal. With the use of carcinoembryonic antigen (CEA) as a

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proof-of-concept analyte, the proposed homogeneous electrochemical aptasensor 6


of the

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exhibits excellent performance in the electrochemical detection of CEA down to the

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80 ag·mL-1 level. This method holds great potential to provide a versatile tool in

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detecting biomolecules in bioanalysis and clinical biomedicine.

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EXPERIMENTAL SECTION

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Chemicals and Materials. The T7Exo was purchased from New England

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Biolabs (Ipswich, MA, USA). All the oligonucleotides were synthesized, HPLC

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purified, and freeze-dried by Shanghai Sangon Biological Engineering Technology

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and Services Co., Ltd. (Shanghai, China). Their sequences are listed in Table S-1. The

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DNA sequences were used as provided and diluted in 10 mM phosphate-buffered

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saline (PBS, pH 7.4), to give stock solutions of 100 µM. CEA was purchased from

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Shanghai lingc-Bio Science Co., Ltd. (Shanghai, China). SYBR Green I was obtained

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from Xiamen Bio-Vision Biotechnology Co. Ltd. (Xiamen, China). NaNO2 and

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4-nitroaniline were purchased from Sigma-Aldrich (Saint Louis, MO, USA). All

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reagents were analytical grade and solutions were prepared using ultrapure water

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(specific resistance of 18 MΩcm). Graphene oxide (GO) was prepared according to

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Hummer’s method55 with small modification, and the detailed characterizations of

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GO was described in Supporting Information.

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Apparatus. All electrochemical experiments were carried out on an Autolab

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electrochemical workstation (Metrohm, Netherland) at room temperature using a

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conventional three-electrode system, whereas the bare or modified GCE electrode

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(CHI104, Φ = 3 mm, CH Instruments, Inc. Shanghai) was used as the working

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electrode, a Ag/AgCl electrode and a platinum wire were employed as the reference 7



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electrode and counter electrode, respectively. The images of gel electrophoresis were

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scanned by the Gel Doc XR+ Imaging System (BIO-RAD, America).

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Fabrication of Reduced GO Modified GCE. The detailed fabrication

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procedures of reduced GO modified GCE (rGO/GCE) are described briefly as follows.

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Prior to the fabrication steps, the GCEs were polished stepwise with aqueous alumina

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slurries of 0.3 and 0.05 µm deagglomerated γ alumina on microcloth, followed by

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rinsing thoroughly with ultrapure water for 30 min. The cleaned GCEs were stored in

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ultrapure water until use. First, an aqueous solution of p-nitrophenyl diazonium salts

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was prepared through dissolving NaNO2 (final concentration: 5 mM) and

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p-nitroaniline (final concentration: 2 mM) in a 0.5 M HCl aqueous solution. The

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mixture was then stirred at dark in ice bath. After 10 min, the obtained solution was

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transferred into the electrochemical cell and the resultant p-nitrophenyl diazonium

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cations were electro-grafted onto the the surface of cleaned bare GCE by sweeping

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two consecutive potential cycles from 0.4 V to −0.2 V (vs Ag/AgCl) at 100 mV·s-1,

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followed by rinsing thoroughly with acetonitrile and ultrapure water to eliminate

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nonspecific adsorption. Then, four potential scans from 0.8 V to −1.2 V (vs Ag/AgCl)

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at 200 mV·s-1 were applied to the as-prepared p-nitrophenyl modified GCE (NP/GCE)

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in 0.25 M H2SO4 solution, followed by electrolysis at a potential of −0.8 V for 60 s to

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ensure all p-nitrophenyl groups had been electrochemically reduced to p-aminophenyl

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groups (AP). The obtained AP-terminated GCE (AP/GCE) were vigorously washed

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with ultrapure water and dried with N2. For preparing rGO/GCE, GO was adsorbed on

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the AP/GCE surface by immersing the AP/GCE in the GO solution (∼0.1 mg·mL-1) 8


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for 6 h. After rinsing with ultrapure water, the reduction of GO on AP/GCE was

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carried out by repetitive potential sweeping from 0.7 V to −1.2 V (vs Ag/AgCl) at a

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scan rate of 50 mV·s-1 in 0.5 M NaCl solution, and then it was thoroughly rinsed with

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ultrapure water. After each step, the electrochemical behavior of the modified GCE

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was characterized by electrochemical impedance spectroscopy (EIS) using

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ferricyanide as redox probe.

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T7Exo-assisted Target-analog Recycling Reaction. Firstly, solutions of HP1

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and HP2 were heated to 95 °C for 3 min and, respectively, allowed to cool to room

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temperature to form the stem-loop DNA structure. Upon optimizing various

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conditions, the analytical procedure for this homogeneous electrochemical aptasensor

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could be briefly described as follows: In this study, the T7Exo-assisted target-analog

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recycling were performed in a 45 µL homogeneous solution consisting of 1.0 µM HP1,

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3.0 µM HP2, 10 U T7Exo in 1× NEBuffer4 (50 mM KAc, 20 mM Tris-Ac, 10 mM

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Mg(Ac)2, 1 mM dithiothreitol, pH 7.9). After the addition of 5.0 µL CEA sample

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solution with different concentrations, the mixture was allowed to react for 100 min at

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25 °C in a constant temperature incubator. The resulting solution (50 µL) was

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transferred onto the rGO/GCE and incubated at room temperature for 30 min,

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followed by thoroughly rinsing with ultrapure water. Finally, the amount of the

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adsorbed MB-ssDNA on rGO/GCE can be detected by performing differential pulse

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voltammetry (DPV) with the potential window from −0.4 to −0.2 V (vs Ag/AgCl) in

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10 mM pH 7.4 PBS. The parameters applied for DPV scanning were 50 ms

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modulation time, 0.5 s interval time, 5 mV step potential, 25 mV modulation 9



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amplitude, and 10 mV·s-1 scan rate. All DPV curves are baseline-corrected using the

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Nova 1.1 software embedded in the Autolab electrochemical workstation.

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Electrophoresis

Demonstration

of

the

Amplification

Strategy.

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Non-denaturating polyacrylamide gel electrophoresis (PAGE) was used to further

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confirm and characterize this novel homogeneous T7Exo-assisted target-analog

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recycling amplification strategy. In the PAGE assay, samples containing different

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DNA structures were added in 1.5 µL 6× loading buffer, respectively. An 8% native

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polyacrylamide hydrogel was prepared using 1× tris-borate-EDTA buffer (TBE, 89

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mM Tris Borate, 2.0 mM EDTA, pH 8.3). The above sample mixtures were injected

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into the polyacrylamide hydrogel for electrophoresis. Electrophoresis was carried out

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at 110 V in TBE buffer for 40 min at room temperature, and stained for 20 min in a

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1×SYBR Green I solution. The resulting gel board was then illuminated with

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ultraviolet light and finally photographed by the gel imaging system.

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RESULTS AND DISCUSSION

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Principle of Affinity-Mediated Homogeneous Electrochemical Aptasensor.

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The working principle of this homogeneous electrochemical aptasensor based on

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T7Exo-assisted target-analog recycling amplification approach for ultrasensitive

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biomolecules detection was illustrated in Scheme 1, and the mechanism of which was

217

verified by non-denaturating PAGE (Figure 1). In our designs, the sequences of HP1

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and HP2 were carefully and rationally designed to kinetically hinder any spontaneous

219

interaction between each other in the absence of target input. As shown in Figure 1,

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both the HP1 (lane-a) and HP2 (lane-b) shows a single and narrow electrophoresis 10


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band, implying that no secondary structure is observed in each rationally designed

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DNA sequence. Although there are complementary domain-b/b* and domain-c/c*

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between HP1 and HP2, mixing the two DNA structures (lane-c) in the absence of

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target CEA does not produce any obvious new band, implying the very low level of

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spontaneous interactions between the rationally designed HP1 and HP2 in the absence

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of target. Furthermore, both HP1 and HP2 could self-hybridize into a stem-loop

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structure with a T7Exo resistant 5’ protruding terminus (Scheme 1). Thus, as

228

illustrated by Figure 1, the T7Exo does not exhibit any activity on the HP1 and HP2

229

in the absence of target (lane-d).

230 231 232

Scheme 1. Schematic illustration of T7Exo-assisted target-analog recycling circuit with isothermal signal amplification ability for this graphene-based homogeneous electrochemical bioassay.

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Moreover, as shown in Table S-1, the designed HP2 consists of a 4 nucleotide (nt)

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5’-protruding ssDNA terminus, 26 base-pair dsDNA stem, and 4 nt ssDNA loop and

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has been modified with a electroactive MB tag at the internal T base of domain-b* in 11



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the stem structure. Due to the short ssDNA domains in HP2, it shows weak and

237

unstable interaction with the rGO/GCE in the absence of target, generating no obvious

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electrochemical signal from the MB tag (Scheme 1). In contrast, when the target CEA

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is introduced to the mixture of HP1, HP2 and T7Exo, CEA could combine with its

240

aptamer region in HP1 to induce a conformational change of HP1 from stem-loop

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structure to CEA@HP1 complex structure (Reaction 1 in Scheme 1), thereby

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exposing the occluded domain-b* and domain-c* in the stem region. As seen from

243

lane-e in Figure 1, the new band, appears at the shorter electrophoresis distance,

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mainly derives from the formation of CEA@HP1 complexes. This result reveals that

245

the designed HP1 could be opened with the recognition of target CEA. In this case,

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the exposed domain-b* and domain-c* could hybridize with HP2 from its

247

5’-protruding terminus to form a blunt 5’-terminus (Reaction 2), whereupon the

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T7Exo catalyzes the stepwise removal of mononucleotides from this protruding

249

domain-b in HP2, liberating CEA@HP1 complex (Reaction 4) before ultimately

250

generating the MB-ssDNA (Reaction 5). As can be expected, the new broad

251

electrophoresis band, displayed in lane-f (Figure 1) with the slowest gel-shift mobility,

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confirms the formation of the CEA@HP1@HP2 complexes. After T7Exo digestion, a

253

new band corresponding to MB-ssDNA (lane-h in Figure 1) is observed in lane-g and

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the brightness of the HP2 band decreased remarkably in lane-g compared with that in

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lane-b, indicated the successful cyclic T7Exo digestion of HP2.

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Importantly, the released CEA@HP1 complex is intact due to its single-strand 5’

257

terminus and will bind another HP2, and the cycle (Reaction 2, 3, 4) starts anew. At 12


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the same time, the originally caged MB-ssDNA in the rigid stem of HP2 is thereby

259

transformed into a flexible MB-ssDNA (Reaction 5) which could hybridize with HP2

260

from its 5’-protruding terminus (Reaction 6) to trigger another cycle of cleavage

261

(Reaction 6, 7, 5). Thus, one target CEA is able to generate a large number of

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MB-ssDNAs, which possess much higher affinity toward the rGO/GCE than HP2

263

(Scheme 1), leading to a distinct increase in the electrochemical signal. The amount of

264

the MB-ssDNA adsorbed on the rGO/GCE surface correlates positively with the

265

concentration of target CEA.

266 267 268 269 270

Figure 1. Non-denaturing PAGE confirmation of the T7Exo-assisted target-analog recycling circuit. Lane-a, HP1; lane-b, HP2; lane-c, HP1+HP2; lane-d, HP1+HP2+T7Exo; lane-e, HP1+CEA; lane-f, HP1+CEA+HP2; lane-g, HP1+CEA+HP2+T7Exo; lane-h, synthetic MB-ssDNA as ssDNA marker.

271

Electrochemical Characterization of rGO/GCE. Figure 2A displays the

272

electrochemical fabrication procedure for rGO/GCE, which employs three

273

electro-reduction steps. In this work, p-nitrophenyl is first electro-grafted onto the

274

GCE surface (Figure 2A-I) by the self-limiting electro-reduction of p-nitrophenyl

275

diazonium salts. The recorded cyclic voltammograms (Figure 2B-I) shows a sharp 13



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irreversible reduction peak at about +0.09 V (vs Ag/AgCl) in the first cycle, which

277

was attributed to the electro-reduction of diazonium cation.56 The disappearance of

278

this reduction peak in the second potential cycle indicated that the electrode surface is

279

passivated due to the formation of self-limiting p-nitrophenyl film. The p-nitrophenyl

280

groups on the GCE were then electrochemically reduced to p-aminophenyl groups

281

(Figure 2A-II). The four cyclic voltammograms were recorded in Figure 2B-II. The

282

large irreversible reduction peak at about -0.65 V in the first scan toward the negative

283

potentials was assigned to electro-reduction of p-nitrophenyl to p-aminophenyl.56 In

284

the subsequent scans, this reduction peak drastically diminishes. After the first

285

scanning, the reversible redox peaks that emerged at about E1/2 = +0.33 V was due to

286

the interconversion between nitrosophenyl and hydroxyaminophenyl.56

287 288 289 290 291 292 293 294

Figure 2. (A) Schematic illustration of the electrochemical fabrication procedure for rGO/GCE, (I) Electro-reduction of p-nitrophenyl diazonium salts; (II) Electro-reduction of p-nitrophenyl groups to p-aminophenyl groups; (III) Electro-reduction of GO to rGO. (B) The recorded electrochemical curves during the electrochemical fabrication of rGO/GCE, (I) Two consecutive CVs of bare GCE in the aqueous solution of p-nitrophenyl diazonium salts; (II) Four consecutive CVs of the as-prepared NP/GCE in 0.25 M H2SO4 solution; (III) Two successive LSVs of GO/GCE in 0.5 M NaCl solution.

295

In this work, GO sheets could be firmly attached onto the AP/GCE surface, 14


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which is mainly ascribed to the π−π stacking interactions between the hexagonal cells

297

of GO and the rings of p-aminophenyl groups as well as the electrostatic attraction

298

between the negatively charged GO sheets and the positively charged p-aminophenyl

299

groups. Finally, rGO/AP/GCE was prepared through electro-reduction of the adsorbed

300

GO on AP/GCE (Figure 2A-III). During the potential scan, an intense reduction peak

301

appeared at about −0.94 V (vs Ag/AgCl) (Figure 2B-III) was observed. It has been

302

reported that this reduction peak came from the reduction of the electroactive oxygen

303

groups on the GO sheets.57 It is notable that the above reduction peak greatly

304

diminished in the subsequent scan, indicated that GO can be completely reduced to

305

rGO sheets in the first scan. Compared with chemical reduction, the electro-reduction

306

approach is green and fast, and does not result in contamination of the obtained rGO.

307

The electrochemical characterization of this sensing platform at each fabrication

308

step is conducted through EIS using [K3Fe(CN)6]/[K4Fe(CN)6] as redox probe. The

309

Nyquist diagrams of the modified GCE at different stages and the corresponding

310

equivalent circuit are displayed in Figure 3A. The EIS include a semicircle at higher

311

frequencies representing the electron-transfer-limited process and a linear part at

312

lower frequencies resulting from the diffusion-limited process. The semicircle

313

diameter equals the interfacial electron-transfer resistance (Ret), which reflects the

314

restricted diffusion of the redox probe accessing the electrode surface. And the Ret

315

values can be obtained from fitted results with the equivalent circuit.

316

As shown in Figure 3A, the bare GCE showed a small semicircle domain (215 Ω,

317

curve a) which indicated the fast electron-transfer process. After the successful 15



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

318

electro-grafting of p-nitrophenyl film on the GCE surface, a remarkable increase in Ret

319

was observed (3080 Ω, curve b), indicating that the formed insulating p-nitrophenyl

320

film suppressed the access of the redox probe to the GCE surface. After

321

electro-reduction, the Ret value of the resulted AP/GCE decreases to 553 Ω (curve c)

322

probably due to the good conductivity of p-aminophenyl, which enhanced the electron

323

transfer between ferricyanide and the GCE. GO/GCE showed a significant increase in

324

Ret value (1730 Ω, curve d), which was mainly attributed to the high electrical

325

resistivity of GO sheets. The electro-reduction of GO sheets resulted in the partially

326

restoration of the sp2-hybridized carbon network to enhance the charge-carrier

327

mobility,58 leading to a remarkable attenuation in Ret (123 Ω, curve e). These EIS

328

measurements demonstrated the successful preparation of rGO/GCE.

329 330 331 332 333 334 335 336

Figure 3. (A) Nyquist plots of the modified GCE at different stages: (a) bare GCE; (b) NP/GCE; (c) AP/GCE; (d) GO/GCE; (e) rGO/GCE. Inset shows magnified EIS spectra. (B) DPV response of rGO/GCE towards the reaction mixture: with no CEA and no T7Exo (a); with 10 U T7Exo and no CEA (b); with 80 ag·mL-1 CEA and no T7Exo (c); with 80 ag·mL-1 CEA and 10 U T7Exo (d); with 0.8 fg·mL-1 CEA and 10 U T7Exo (e); containing HP3 as a substitute of HP2 in the presence of 0.8 fg·mL-1 CEA and 10 U T7Exo (f). These DPV curves are collected in 10 mM pH 7.4 PBS. Inset shows magnified DPV curves.

337

Feasibility of this Homogeneous Electrochemical Aptasensor. DPV is utilized

338

to further verify the feasibility of the designed T7Exo-assisted target-analog recycling

339

amplification process for this homogeneous electrochemical biosensor using 16


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340

rGO/GCE as the sensing platform in the presence/absence of target with/without

341

T7Exo. As depicted in Figure 3B, in the absence of both target and T7Exo (curve a),

342

the oxidation peak current of MB is very low (curve a), indicating the weak affinity

343

between HP2 and rGO/GCE. In the absence of target with the addition of T7Exo (10

344

U), a negligible DPV response was observed (curve b), which could be ascribed not

345

only to the ultralow background reactivity between HP1 and HP2 in the absence of

346

target, but also to the ultralow cleavage activity of T7Exo toward the designed HP1

347

and HP2 in the absence of target. Furthermore, in the presence of target (80 ag·mL-1)

348

without the addition of T7Exo (curve c), no obvious change in peak current was

349

obtained compared to curve b and curve a, suggesting that the T7Exo-assisted cyclic

350

cleavage of HP2 could be only started in the presence of both target and T7Exo. As

351

expected, a large and well-defined oxidation peak around −0.3 V can be observed in

352

the presence of both 80 ag·mL-1 CEA and 10 U T7Exo (curve d). This signal increase

353

is contributed to the electrochemical oxidation of the MB tags in MB-ssDNAs, which

354

were generated from the T7Exo-assisted cyclic cleavage of HP2 and adsorbed stably

355

on the electrode surface due to its higher affinity toward rGO/GCE. Moreover, the

356

remarkable peak current improvement obtained from the increased concentration of

357

CEA (curve e) illustrates the high sensitivity of the proposed method.

358

Subsequently, we carried out a control experiment to further prove the signal

359

amplification effect of the designed T7Exo-assisted target-analog recycling strategy.

360

Toward this goal, another MB-labeled hairpin DNA reporter (HP3) was designed as a

361

substitute of HP2 for comparison. As shown in Table S-1, the difference between HP3 17



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

362

and HP2 only lies in that the domain-b* sequences in the stem region of HP2 are

363

changed to domain-h*, which is non-complementary to domain-b. That is, the

364

recognition of CEA with HP1 could also trigger the cleavage process of T7Exo

365

toward HP3 and release the CEA@HP1 complex to trigger the successive cleavage

366

process, but the finally generated MB-ssDNA containing domain-h* could not

367

hybridize with HP3 again to initiate another cycle of cleavage, resulting a typical

368

target recycling amplification process. As expected, the DPV response with this

369

typical target recycling amplification (curve f) was much lower than that with

370

T7Exo-assisted target-analog recycling amplification (curve e) under the same

371

experimental conditions. Such greatly enhanced current in curve e basically affirms

372

that much more MB-ssDNAs could be generated in the T7Exo-assisted target-analog

373

recycling amplification process, demonstrating the feasibility of the designed

374

T7Exo-assisted target-analog recycling strategy for this homogeneous electrochemical

375

biomolecule detection.

376

Analytical Performance. The feasibility of this homogeneous electrochemical

377

biosensor described above suggests that this T7Exo-assisted target-analog recycling

378

amplification strategy could be utilized for the amplified biomolecules detection with

379

ultrahigh sensitivity. We challenged this homogeneous electrochemical biosensor with

380

target CEA of different concentrations to test its analytical performance under the

381

optimal conditions (Detail optimizations are shown in Supporting Information).

382

Figure 4A illustrates the DPV responses observed upon different concentrations of

383

CEA. It is clearly shown that the DPV peak current increased significantly with 18


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384

elevated concentrations of target CEA. This increase could be attributed to the fact

385

that the presence of more target CEA caused the generation of more MB-ssDNAs,

386

after which the single-stranded MB-ssDNAs are adsorbed on the rGO/GCE surface,

387

enhancing the peak current. A calibration curve made by plotting ∆I (∆I = I − I0; I is

388

the current of target at different concentrations; the current response is recorded as I0

389

when the target concentration was zero) versus the target CEA concentration is shown

390

in Figure 4B. It can be clearly seen that ∆I is proportional to the logarithmic (lg) value

391

of the target CEA concentration over a 4 orders of magnitude range from 80 ag·mL-1

392

to 950 fg·mL-1. The resulting linear regression equations were obtained as ∆I(nA) =

393

212.43lg[cCEA(fg·mL-1)]+247.16 with a correlation coefficient of 0.9976, Additionally,

394

the directly measured detection limit is 80 ag·mL-1, which is much lower than that of

395

the existing signal amplification strategies applied for CEA quantification, such as

396

MNAzyme catalyzed cyclic cleavage of DNA hairpin probe (2.0 pg·mL-1),59

397

amplification based on hybridization chain reaction (HCR, 10 pg·mL-1),60 and

398

quadruple signal amplification based on adivin−biotin chemistry, gold nanoparticles,

399

HCR, and DNAzyme (1.0 fg·mL-1).61 More and detailed comparisons of the analytical

400

performance with previously reported methods are shown in Table S-2. These results

401

further demonstrate the amplification efficiency of T7Exo-assisted target-analog

402

recycling strategy and reveal that the proposed biosensor is efficient for ultrasensitive

403

detection of CEA.

19



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

404 405 406 407 408 409

Figure 4. (A) DPV responses of the proposed homogeneous electrochemical biosensor to different concentrations of CEA (from a to r: 0, 0.08, 0.1, 0.3, 0.5, 0.8, 1.0, 3.0, 5.0, 8.0, 10, 30, 50, 80, 100, 300, 500 and 950 fg·mL-1). (B) Variation of the peak current value as a function of CEA concentration. Inset: the linear relationship of ∆I versus the logarithm of CEA concentration. The error bars represent the standard deviations of eleven parallel tests.

410

Selectivity, Reproducibility, and Stability. To investigate the selectivity and

411

specificity of the proposed homogeneous electrochemical aptasensor, we performed a

412

series of contrast experiments using other protein biomarkers at the same

413

concentration as interfering substances, such as alpha-fetoprotein (AFP), carcinoma

414

antigen 125 (CA125), carcinoma antigen 199 (CA199). As shown in Figure 5, the

415

contrast experiments were performed by assaying AFP (1.0 pg·mL-1), CA125 (1.0

416

pg·mL-1), and CA199 (1.0 pg·mL-1) instead of CEA (80 ag·mL-1), respectively, at the

417

same optimal conditions. We can see that only CEA resulted in an evident DPV

418

response, the peak current value of AFP, CA125, and CA199 did not exhibit any

419

obvious increase compared with the blank. Moreover, an 80 ag·mL-1 CEA sample

420

coexisted with 1.0 pg·mL-1 AFP, 1.0 pg·mL-1 CA125, and 1.0 pg·mL-1 CA199 does

421

not exhibit obvious peak current increase compared with that obtained from 80

422

ag·mL-1 CEA only.

20


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423 424 425 426

Figure 5. Selectivity of this electrochemical biosensor. (A) Blank; (B) 80 ag·mL-1 CEA; (C) 1.0 pg·mL-1 AFP; (D) 1.0 pg·mL-1 CA125; (E) 1.0 pg·mL-1 CA199; (F) 80 ag·mL-1 CEA sample coexisted with 1.0 pg·mL-1 AFP, 1.0 pg·mL-1 CA125, and 1.0 pg·mL-1 CA199.

427

The reproducibility of the present biosensor was investigated with inter-assay

428

coefficient of variation (%). The inter-assay precision was assessed by assaying the

429

same CEA sample with ten different biosensors prepared independently at the same

430

experimental conditions. The relative standard deviations for the parallel detection of

431

80 ag·mL-1, 5 fg·mL-1, and 500 fg·mL-1 CEA with 10 different biosensors,

432

respectively, were 4.39%, 3.66%, and 3.54%. Besides reproducibility, stability of the

433

biosensors was also investigated. When the biosensor was stored in dry at 4 °C, over

434

98.3% of the initial response was remained after a storage period of 2 weeks,

435

indicating the proposed biosensor possessed acceptable stability. These evaluations

436

suggest a great potential of this homogeneous electrochemical biosensor for the

437

monitoring of CEA in clinical sample for early diagnosis of cancers.

438

Real Sample Analysis. A preliminary application of the proposed homogeneous

439

electrochemical aptasensor in real samples was performed through the measurements

440

of five human serum samples to evaluate the analytical reliability and application

441

potential of the proposed method. When the levels of CEA concentrations in the 21



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Page 22 of 29

442

human serum samples are over the upper limit of the calibration range, the human

443

serum samples were appropriately diluted with 10 mM pH 7.4 PBS prior to assay. The

444

assay results obtained by this method were compared with those obtained by the

445

commercially used automated electrochemiluminescent immunoassay analyzer

446

(Elecsys 2010, Roche) in cancer hospitals. The results were shown in Table 1. The

447

proposed biosensing strategy showed an acceptable agreement with the reference

448

method. These results suggest that this proposed homogeneous electrochemical

449

aptasensor may be competent for monitoring real samples.

450

Table 1. Assay results of real human serum by the proposed and reference method Samples Sample-1 Sample-2 Sample-3 Sample-4 Sample-5

CEA concentration (ng·mL-1) Proposed method*

Reference method

Relative error (%)

54.47 33.51 9.94 22.36 17.49

53.49 34.21 9.56 22.97 11.16

1.83 -2.06 3.94 -2.65 2.97

451

*Average of eleven measurements.

452

CONCLUSIONS

453

In summary, we have developed a novel affinity-mediated electrochemical

454

biosensing platform that is able to carry out ultrasensitive detection of target

455

biomolecule in a homogeneous solution without immobilization of any bioprobe on

456

electrode surface. rGO/GCEs, equipped with convenient and “green” fabrication route,

457

excellent conductivity, stability, and reproducibility, performed as an appropriate

458

electrochemical sensing platform for this homogeneous electrochemical aptasensor. It

459

is found that rGO/GCEs are able to effectively differentiate the designed HP2 and

460

MB-ssDNA owing to the different affinities of ssDNA and dsDNA structure to

461

graphene. On the basis of this unique property of rGO/GCE, we have developed a 22


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462

novel single-step T7Exo catalyst DNA digestion circuit, in which the specific

463

recognition of target biomolecule triggers on the numerous transformation of HP2 to

464

MB-ssDNA. Furthermore, the amplified detection of target biomolecule is realized

465

through the recycling of the two target-analogs, e.g. MB-ssDNA and target@HP1,

466

simultaneously. With the use of CEA as a proof-of-concept target, our proposed

467

sensor exhibits ultra-sensitivity with a directly measured detection limit of 80 ag·mL-1,

468

which is lower than most previously reported CEA biosensors. The versatility of this

469

homogeneous sensing platform to detect aptamer substrates or DNA allows the rapid

470

design of sensing systems for numerous target molecules. We envision that the

471

homogeneous electrochemical approach described herein will find useful applications

472

in the biological, medical, and environmental fields.

473

ASSOCIATED CONTENT

474

Supporting Information

475

Sequences of oligonucleotides used in this work, transmission electron microscope

476

and atomic force microscope characterizations of GO, detailed optimizations of the

477

experimental conditions, and comparison of the present study with other sensing

478

strategies. This material is available free of charge via the Internet at

479

http://pubs.acs.org.

480

Notes

481 482 483

The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was financially supported by National Natural Science Foundation of

23



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484

China (21545005, 31501570, 21375072), Natural Science Foundation of Shandong

485

Province, China (ZR2014BQ011), Research Foundation for Distinguished Scholars of

486

Qingdao Agricultural University (663-1115003, 663-1113311), and the Special

487

Foundation for Taishan Scholar of Shandong Province.

488

REFERENCES

489

(1) Heller, M. J. Annu. Rev. Biomed. Eng. 2002, 4, 129–153.

490

(2) McGlennen, R. C. Clin. Chem. 2001, 47, 393–402.

491

(3) Li, W.; Zhang, Q.; Zhou, H.; Chen, J.; Li, Y.; Zhang, C.; Yu, C. Anal. Chem. 2015,

492

87, 8336–8341.

493

(4) Zong, C.; Wu, J.; Liu, M.; Yang, L.; Liu, L.; Yan, F.; Ju, H. Anal. Chem. 2014, 86,

494

5573–5578.

495

(5) Xia, H.; Li, L.; Yin, Z.; Hou, X.; Zhu, J.-J. ACS Appl. Mater. Interfaces 2015, 7,

496

696–703.

497

(6) Jie, G.; Yuan, J. Anal. Chem. 2012, 84, 2811–2817.

498

(7) Li, L.; Wang, Q.; Feng, J.; Tong, L.; Tang, B. Anal. Chem. 2014, 86, 5101–5107.

499

(8) Li, J.; Zhong, X.; Zhang, H.; Le, X. C.; Zhu, J.-J. Anal. Chem. 2012, 84,

500

5170–5174.

501

(9) Wang, C.; Qian, J.; Wang, K.; Wang, K.; Liu, Q.; Dong, X.; Wang, C.; Huang, X.

502

Biosens. Bioelectron. 2015, 68, 783–790.

503

(10) Li, C.; Wang, H.; Shen, J.; Tang, B. Anal. Chem. 2015, 87, 4283–4291.

504

(11) Golub, E.; Pelossof, G.; Freeman, R.; Zhang, H.; Willner, I. Anal. Chem. 2009, 81,

505

9291–9298. 24


Page 24 of 29

Page 25 of 29

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


506

(12) Zheng, T.; Zhang, Q.; Feng, S.; Zhu, J.-J.; Wang, Q.; Wang, H. J. Am. Chem. Soc.

507

2014, 136, 2288–2291.

508

(13) Zhu, C.; Yang, G.; Li, H.; Du, D.; Lin, Y. Anal. Chem. 2015, 87, 230–249.

509

(14) Zhuang, J.; Tang, D.; Lai, W.; Chen, G.; Yang, H. Anal. Chem. 2014, 86,

510

8400–8407.

511

(15) Dong, S.; Zhao, R.; Zhu, J.; Lu, X.; Li, Y.; Qiu, S.; Jia, L.; Jiao, X.; Song, S.; Fan,

512

C.; Hao, R.; Song, H. ACS Appl. Mater. Interfaces 2015, 7, 8834–8842.

513

(16) Shi, J.-J.; He, T.-T.; Jiang, F.; Abdel-Halim, E. S.; Zhu, J.-J. Biosens. Bioelectron.

514

2014, 55, 51–56.

515

(17) Zhang, X.; Chen, C.; Yin, J.; Han, Y.; Li, J.; Wang, E. Anal. Chem. 2015, 87,

516

4612–4616.

517

(18) Xiang, Y.; Lu, Y. Nat. Chem. 2011, 3, 697–703.

518

(19) Cao, J.-T.; Hao, X.-Y.; Zhu, Y.-D.; Sun, K.; Zhu, J.-J. Anal. Chem. 2012, 84,

519

6775–6782.

520

(20) Du, D.; Wang, J.; Lu, D.; Dohnalkova, A.; Lin, Y. Anal. Chem. 2011, 83,

521

6580–6585.

522

(21) Rowe, A. A.; Chuh, K. N.; Lubin, A. A.; Miller, E. A.; Cook, B.; Hollis, D.;

523

Plaxco, K. W. Anal. Chem. 2011, 83, 9462–9466.

524

(22) Ricci, F.; Lai, R. Y.; Heeger, A. J.; Plaxco, K. W.; Sumner, J. J. Langmuir 2007,

525

23, 6827–6834.

526

(23) Degliangeli, F.; Kshirsagar, P.; Brunetti, V.; Pompa, P. P.; Fiammengo, R. J. Am.

527

Chem. Soc. 2014, 136, 2264–2267. 25



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

528

(24) Prigodich, A. E.; Alhasan, A. H.; Mirkin, C. A. J. Am. Chem. Soc. 2011, 133,

529

2120–2123.

530

(25) Wei, X.; Ma, X.; Sun, J. J.; Lin, Z.; Guo, L.; Qiu, B.; Chen, G. Anal. Chem. 2014,

531

86, 3563–3567.

532

(26) Xuan, F.; Luo, X.; Hsing, I. M. Anal. Chem. 2013, 85, 4586–4593.

533

(27) Xuan, F.; Luo, X.; Hsing, I. M. Anal. Chem. 2012, 84, 5216–5220.

534

(28) Luo, X.; Lee, T. M.-H.; Hsing, I. M. Anal. Chem. 2008, 80, 7341–7346.

535

(29) Xuan, F.; Luo, X.; Hsing, I. M. Biosens. Bioelectron. 2012, 35, 230–234.

536

(30) Nie, J.; Zhang, D.-W.; Zhang, F.-T.; Yuan, F.; Zhou, Y.-L.; Zhang, X.-X. Chem.

537

Commun. 2014, 50, 6211–6213.

538

(31) Liu, B.; Zhang, B.; Chen, G.; Tang, D. Chem. Commun. 2014, 50, 1900–1902.

539

(32) Wang, Y.; Li, Z.; Wang, J.; Li, J.; Lin, Y. Trends Biotechnol. 2011, 29, 205–212.

540

(33) Lu, C.-H.; Yang, H.-H.; Zhu, C.-L.; Chen, X.; Chen, G.-N. Angew. Chem. Int. Ed.

541

2009, 48, 4785–4787.

542

(34) He, S.; Song, B.; Li, D.; Zhu, C.; Qi, W.; Wen, Y.; Wang, L.; Song, S.; Fang, H.;

543

Fan, C. Adv. Funct. Mater. 2010, 20, 453–459.

544

(35) Huan, J.; Liu, Q.; Fei, A.; Qian, J.; Dong, X.; Qiu, B.; Mao, H.; Wang, K. Biosens.

545

Bioelectron. 2015, 73, 221–227.

546

(36) Walch, N. J.; Davis, F.; Langford, N.; Holmes, J. L.; Collyer, S. D.; Higson, S. P.

547

J. Anal. Chem. 2015, 87, 9273–9279.

548

(37) Li, G.; Yu, X.; Liu, D.; Liu, X.; Li, F.; Cui, H. Anal. Chem. 2015, 87,

549

10976–10981. 26


Page 26 of 29

Page 27 of 29

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


550

(38) Lin, Y.; Zhou, Q.; Lin, Y.; Tang, D.; Niessner, R.; Knopp, D. Anal. Chem. 2015,

551

87, 8531–8540.

552

(39) Fei, A.; Liu, Q.; Huan, J.; Qian, J.; Dong, X.; Qiu, B.; Mao, H.; Wang, K. Biosens.

553

Bioelectron. 2015, 70, 122–129.

554

(40) Liu, H.; Li, L.; Wang, Q.; Duan, L.; Tang, B. Anal. Chem. 2014, 86, 5487–5493.

555

(41) Zhang, H.; Jia, S.; Lv, M.; Shi, J.; Zuo, X.; Su, S.; Wang, L.; Huang, W.; Fan, C.;

556

Huang, Q. Anal. Chem. 2014, 86, 4047–4051.

557

(42) Wang, Y.; Li, Z.; Hu, D.; Lin, C.-T.; Li, J.; Lin, Y. J. Am. Chem. Soc. 2010, 132,

558

9274–9276.

559

(43) Zhang, H.; Li, F.; Dever, B.; Li, X.-F.; Le, X. C. Chem. Rev. 2013, 113,

560

2812–2841.

561

(44) Ye, S.; Wu, Y.; Zhai, X.; Tang, B. Anal. Chem. 2015, 87, 8242–8249.

562

(45) Zhang, Y.; Hu, J.; Zhang, C. Y. Anal. Chem. 2012, 84, 9544–9549.

563

(46) Wang, F.; Freage, L.; Orbach, R.; Willner, I. Anal. Chem. 2013, 85, 8196–8203.

564

(47) Liu, X.; Freeman, R.; Willner, I. Chem. Eur. J. 2012, 18, 2207–2211.

565

(48) Liu, S.; Wang, Y.; Zhang, C.; Lin, Y.; Li, F. Chem. Commun. 2013, 49,

566

2335–2337.

567

(49) Zhu, X.; Zhao, J.; Wu, Y.; Shen, Z.; Li, G. Anal. Chem. 2011, 83, 4085–4089.

568

(50) Wu, D.; Xin, X.; Pang, X.; Pietraszkiewicz, M.; Hozyst, R.; Sun, X. g.; Wei, Q.

569

ACS Appl. Mater. Interfaces 2015, 7, 12663–12670.

570

(51) Duan, R.; Zuo, X.; Wang, S.; Quan, X.; Chen, D.; Chen, Z.; Jiang, L.; Fan, C.;

571

Xia, F. J. Am. Chem. Soc. 2013, 135, 4604–4607. 27



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

572

(52) Wang, M.; Fu, Z.; Li, B.; Zhou, Y.; Yin, H.; Ai, S. Anal. Chem. 2014, 86,

573

5606–5610.

574

(53) Zhu, G.; Liang, L.; Zhang, C. Y. Anal. Chem. 2014, 86, 11410–11416.

575

(54) Kerr, C.; Sadowski, P. D. J. Biol. Chem. 1972, 247, 305–310.

576

(55) Hummers, W. S.; Offeman, R. E. J. Am. Chem. Soc. 1958, 80, 1339–1339.

577

(56) Brooksby, P. A.; Downard, A. J. Langmuir 2004, 20, 5038–5045.

578

(57) Wang, Z.; Zhou, X.; Zhang, J.; Boey, F.; Zhang, H. J. Phys. Chem. C 2009, 113,

579

14071–14075.

580

(58) Park, S.; Ruoff, R. S. Nat. Nanotechnol. 2009, 4, 217–224.

581

(59) Ren, K.; Wu, J.; Ju, H.; Yan, F. Anal. Chem. 2015, 87, 1694–1700.

582

(60) Xu, J.; Wu, J.; Zong, C.; Ju, H.; Yan, F. Anal. Chem. 2013, 85, 3374–3379.

583

(61) Zhou, J.; Lai, W.; Zhuang, J.; Tang, J.; Tang, D. ACS Appl. Mater. Interfaces

584

2013, 5, 2773–2781.

28


Page 28 of 29

Page 29 of 29

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585

For TOC only

586

29


Affinity-Mediated Homogeneous Electrochemical Aptasensor on a

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