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Enzymatic repairing amplification-based versatile signal-on fluorescence sensing platform for detecting pathogenic bacteria
Accepted Manuscript Title: Enzymatic Repairing Amplification-based Versatile Signal-on Fluorescence Sensing Platform for Detecting Pathogenic Bacteria Author: Xueqi Leng Yu Wang Su Liu Qianqian Pei Xuejun Cui Yuqin Tu Xuejiao Liu Jiadong Huang PII: DOI: Reference:
S0925-4005(17)30915-2 http://dx.doi.org/doi:10.1016/j.snb.2017.05.092 SNB 22376
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
11-3-2017 16-5-2017 17-5-2017
Please cite this article as: X. Leng, Y. Wang, S. Liu, Q. Pei, X. Cui, Y. Tu, X. Liu, J. Huang, Enzymatic Repairing Amplification-based Versatile Signal-on Fluorescence Sensing Platform for Detecting Pathogenic Bacteria, Sensors and Actuators B: Chemical (2017), http://dx.doi.org/10.1016/j.snb.2017.05.092 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.
Enzymatic Repairing Amplification-based Versatile Signal-on
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Fluorescence Sensing Platform for Detecting Pathogenic
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Bacteria
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Xueqi Lenga, Yu Wangb, Su Liua*, Qianqian Peic, Xuejun Cuic, Yuqin Tua, Xuejiao Liub, Jiadong
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Huangb,c
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a College of Resources and Environment, University of Jinan, Jinan 250022, P. R. China
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b College of Biological Sciences and Technology, University of Jinan, Jinan 250022, P. R. China
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c Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P. R. China.
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Corresponding author. E-mail: [email protected]; Tel.: +86-531-89736122; Fax: +86-531-
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Abstract A novel fluorescence biosensing strategy for ultrasensitive and specific detection of pathogenic
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bacteria based on target-triggered enzymatic repairing amplification (ERA) has been developed. This
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strategy relies on target-aptamer binding mediated ERA reaction, which is carried out cyclically with
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the help of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and
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endonuclease IV (Endo IV) to produce amplified fluorescence signal. In our assay, the specially
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designed hairpin probe (HAP) is used as DNA template responsible for producing a great quantity of
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reporter oligonucleotides and secondary primers, which can initiate a new cycle of
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polymerization-repairing amplification. Moreover, by the combination of polymerase-catalyzed
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incorporation of lesion bases with UDG and Endo IV-assisted ERA, multiple cycle of amplification
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of the recognition event is achieved, enabling ultrasensitive detection of pathogenic bacteria. Under
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optimal conditions, this biosensor exhibits ultrasensitivity toward target pathogenic bacteria with
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detection limits of 9.86 cfu mL-1 and a detection range of 5 orders of magnitude. Additionally, the
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biosensor has the ability of combating nonspecific background. Furthermore, an archer probe
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containing the anti-target aptamer sequence and a primer sequence is designed, which translates the
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binding of target to aptamer into the presence of primer sequence, enabling the detection of various
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targets, such as protein, DNA, small molecular, and any substance possessing its aptamer. Hence, the
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proposed target-triggered ERA-based signal-on fluorescence sensing strategy indeed create a
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versatile and useful platform for detection of pathogenic bacteria, related food safety analysis and
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clinical diagnosis.
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Keywords: signal-on fluorescence sensing; homogeneous detection; pathogenic bacteria;
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target-triggered enzymatic repairing amplification
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1. Introduction Foodborne pathogenic bacteria have always been a major threat to human health. In 2011, the
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U.S. Centers for Disease Control and Prevention reported the damage caused by foodborne diseases
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each year in the United States: they caused 48 million people to fall sick, 128 000 to be hospitalized,
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and 3000 to die [1,2]. What’s more, the situation in developing countries is even more serious.
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Besides, the infectious dose of most pathogenic bacteria is as low as 10 colony-forming units (cfu)
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[3]. Thus, sensitive, robust, low cost, and ready-to-use analysis of pathogenic bacteria represents a
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critical approach to prevent and control foodborne diseases.
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Currently, the gold standard for bacterial detection remains the culture method. Though great
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progress in sensitivity and specificity have been made over the years, including the incorporation of
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chromogenic agars, the culture approach still suffers from time-consuming for routine analysis in the
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food industry. Culture methods require several days for pre-enrichment, enrichment, selective plating,
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identification, and confirmation, at which point a contaminated product could have already reached
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the consumer [4,5]. Thus, the standard culture methods are neither quick nor simple, making their
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use at the processing plant level cumbersome and limited.
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In recent years, various biosensing technologies, including electrochemical method [6,7,8],
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electrogenerated chemiluminescence assay [9] and colorimetric analysis [10,11], have been
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developed to improve the sensitivity and specificity, reduce cost and shorten analysis time in
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foodborne pathogenic bacteria analysis. Electrochemical-based assay method possesses the
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advantages of high sensitivity, but its accuracy and reproductivity are far from ideal.
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Electrogenerated chemiluminescence biosensor has the advantages of high sensitivity and ease of
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control, but it isn’t a versatile platform for analyst. Colorimetric method offers the ability for visual
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detection, and high portability, however, it hasn’t satisfactory sensitivity. Thereby, it’s still a
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challenge to develop simple, rapid, low cost and high sensitive pathogenic bacteria assay methods. There are two factors crucial for affecting the performance of the biosensor, namely specificity
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and sensitivity. In order to improve the sensitivity in the detection of pathogenic bacteria, abundant
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nucleic acid amplification technologies is increasingly developed, such as reverse transcription
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quantitative
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amplification [18,19,20], loop mediated isothermal amplification [21,22,23], hybridization chain
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reaction [24,25,26]. Most of these amplification reactions involve a key mechanism of
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“cleavage-replication” cyclical reaction, in which a phosphodiester bond is cleaved cyclically by
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nicking endonuclease such that 3′ end at the nick site can act as a primer to initiate a new cycle of
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extension reaction with the aid of polymerase. However, it is reported that the combination of
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nicking endonuclease and DNA polymerase may cause nonspecific amplification even in the absence
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of DNA template [27, 28], possibly leading to false positive signal in the detection. Thus, it is of
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great significance to develop alternative mechanisms that is capable of efficient amplification via
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polymerase with low background.
rolling circle
amplification [15,16,17],
strand-displacement
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PCR [12,13,14],
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Herein, we report the development of a novel fluorescence biosensing strategy for ultrasensitive
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and highly specific detection of pathogenic bacteria based on target-triggered enzymatic repairing
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amplification (ERA). The ERA reaction is initiated specifically by target pathogens-aptamer binding
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and carried out cyclically with the aid of polymerase and two DNA repairing enzymes, uracil-DNA
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glycosylase (UDG) and endonuclease IV (Endo IV) to produce amplified fluorescence signal. To the
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best of our knowledge, this is the first time that the target-aptamer binding triggered ERA has been
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utilized for fluorescence sensing for pathogenic bacteria. Our strategy features with several
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significant aspects. First, a hairpin probe (HAP) is designed to function as DNA template responsible
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for producing a great quantity of reporter oligonucleotides and secondary primers, which can anneal
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with another HAP and initiate a new cycle of polymerization-repairing amplification. Moreover, the
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fluorescence-quenched probe can cyclically be cleaved via Endo IV-catalyzed repairing reaction.
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Thus, multiple cycle of amplification of the recognition event is achieved by the combination of
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polymerase-catalyzed incorporation of lesion bases with UDG and Endo IV-assisted ERA, enabling
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ultrasensitive quantity of pathogenic bacteria. Second, the results reveal the developed ERA-based
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biosensing strategy could combat nonspecific background, which is probably attributed that
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nonspecific extension of nucleotides might incorporate too many lesions to grow into long DNA
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replicates. Third, the specialized design of an archer probe containing the anti-target aptamer
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sequence and a universal primer sequence translates the binding of target to aptamer into the
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presence of primer sequence, which enables the detection of various targets, such as protein, DNA,
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small molecular, and any substance possessing its aptamer. Therefore, the target-triggered
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ERA-based signal-on fluorescence sensing strategy indeed creates a versatile and useful platform for
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ultrasensitive and highly specific assay of pathogenic bacteria.
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2. Materials and methods
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2.1. Materials and reagents
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All oligonucleotides were HPLC-Purified and synthesized by Sangon Biotechnology Co. Ltd.
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(Shanghai, China). The sequences of oligonucleotides are listed in Table 1. E.coli uracil DNA
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glycosylase (UDG), endonuclease IV (Endo IV) and Bst DNA polymerase (large fragment) were
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purchased from New England Biolabs (Ipswich, MA, USA). dATP, dGTP, dCTP, dUTP, and dTTP
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were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Salmonella Typhimurium
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(KCTC 1925), E. coli (KCTC 2571), Bacillus subtilis (KCTC 1028) and Listeria (KCTC 3569) were
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obtained from Institute of Microbiology Chinese Academy (Beijing, China). Peptone, beef extract
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powder, bacto-tryptone and bacto-yeast extract were obtained from Qingdao Biological Technology
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Co. Ltd. (Qingdao, China). The agarose and DNA marker were purchased by Takara Biotechnology
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Co. Ltd. (Dalian, China). Other chemicals were of analytical grade and were used without further
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purification. Ultrapure water obtained from a Millipore filtration system was used throughout.
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Tab. 1 DNA Oligonucleotides Sequence Used in This Work
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sequence (5’ to 3’) description
Apt
AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA
P1
TCTTTTCCAAAACGGGCATTACT
HAP
GCCTCCGCTTGAGCCCTGTGTCCCGTCATGCGAGTAATGCCCAAA
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fluorescence-quenched
GCCT(FAM)XCGC(Dabcyl)TTG
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probe The underlined bases in Apt are matched bases with P1. Three regions in HAP are marked in
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three different colors. The primer annealing region (P1*) is highlighted in blue, the secondary primer
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annealing region (P2*) is highlighted in red, the reporter coding region (R*) is highlighted in green.
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The fluorescence-quenched probe is with 4-(4-dimethylaminophenyl) diazenylbenzoic acid (Dabcyl)
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quencher and fluorescein isothiocyanate (FAM) fluorophore, X is the tetrahydrofuran abasic site
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mimic.
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2.2 Apparatus
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Gel electrophoresis was conducted using DYCZ-24DN electrophoresis cell (LIUYI, Beijing,
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China) and Bio-Rad Gel imaging system (Bio-Rad, USA). Fluorescence spectra were recorded using
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a Agilent Cary Eclipse spectrofluorometer (Hitachi, Japan) with excitation at 494 nm. The slit was
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set to be 5 nm for both the excitation and the emission. 6
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2.3 Bacterial Strains and Growth Conditions. All bacteria were freshly harvested from the bacterial culture after incubation at 37 °C for 12 h.
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The resulting bacterial samples were centrifuged at 800 rpm for 5 min. The supernatant was removed.
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The bacterial cells were rinsed with PBS solution (10 mM, pH 7.4) through centrifugation at 800 rpm
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for 5 min. The obtained bacterial cells were resuspended in PBS solution (10 mM, pH 7.4). The
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bacterial number was determined by the bacteria chamber.
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2.4 S. Typhimurium detection procedures
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All samples were prepared in 4 µL Tris-HCl reaction buffer (50mM, pH 7.9 containing 100 mM
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NaCl, 10 mM MgSO2) for the detection of S. Typhimurium. The detailed procedure was as follows:
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Firstly, Apt (3 µL, 10 µM ) and P1 (3 µL, 10 µM) were mixed at 37 ℃ for 30 min and stored at -20
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ºC until use. Subsequently, HAP (6 µL, 10 µM), fluorescence-quenched probe (3 µL, 20 µM) and
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equal volumes of different concentrations of S. Typhimurium (or other foodborne diseases), Bst DNA
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polymerase (2 µL, 8000 U/mL), UDG (2.4 µL, 10 U/µL), Endo IV (2.4 µL, 20 U/µL), 10 mM dNTPs
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(10 mM dATP, dGTP, dCTP, dUTP each) were mixed at 50 ℃ for 90 min. At last, the above mixed
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solution (30 µL) was mixed with 120 µL of ultrapure water and scanned using Agilent Cary Eclipse
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spectrofluorometer. Unless noted otherwise, all experiments for S. Typhimurium measurements were
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repeated three times in this study.
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2.5 Gel electrophoresis
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The gel electrophoresis was performed using the DNA samples (5 µL per well) on a 4% agarose
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gel with a fluorescence stain Cyber Gold (0.5 µg/ mL) in 50 mM Tris-borate running buffer (pH 8.2)
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containing 2 mM EDTA at 145 V for 30 min. After electrophoresis, the gel was visualized via
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fluorescence detection using a Bio-Rad Gel imaging system.
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3. Results and Discussion
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3.1 Principle of the ERA-based versatile fluorescence sensing strategy for ultrasensitive detection of
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pathogenic bacteria
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Scheme 1 illustrates the working principle of the fluorescence sensing strategy for ultrasensitive
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and highly specific assay of pathogenic bacteria based on target-aptamer binding triggered ERA, and
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S. Typhimurium is selected as a model analyte. An arched probe is designed to contain the S.
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Typhimurium aptamer sequence and a primer (P1) sequence, which is complementary to a DNA
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segment (P1*) at the 3’ end of the hairpin probe (HAP). The HAP consists of three regions and two
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lesion incorporating sites: a primer annealing region (P1*), a secondary primer annealing region
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(P2*), and a reporter coding region (R*) that is used to produce reporter oligonucleotides
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complementary to the fluorescence-quenched probe. The fluorescence-quenched probe is designed to
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have a tetrahydrofuran abasic site mimic flanked in proximity to nucleotides labelled with a
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fluorophore (FAM) and a quencher (Dabcyl), which is complementary to the reporter oligonucleotide
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(R). In the presence of target S. Typhimurium, the arched probe specifically combines to S.
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Typhimurium, which leads to the release of P1. Then P1 hybridizes with HAP and functions as a
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primer to initiate an extension reaction and replicates the DNA template in HAP with the aid of Bst
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DNA polymerase and four nucleotides dATP, dGTP, dCTP and dUTP. It is worth noting that dUTP is
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used to instead of dTTP to incorporate lesions. So, two uracil (dU) nucleotides are incorporated in
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the extension product. Immediately, the two lesion sites are excised by UDG-catalyzed hydrolysis
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reaction, which results in the N-glycosylic bond joining the uracil base to deoxyribose. The produced
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abasic sites (AP site) are subsequently cleaved by Endo IV at the phosphodiester bond upstream to
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the AP site, which generates a secondary primer (P2) and reporter oligonucleotide R. Then, a new
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cycle of "extension-excision-cleavage" reaction occurs with the help of polymerase, UDG, and Endo
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IV, producing a large number of copies of secondary primer P2 and reporter oligonucleotide R.
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Meanwhile, the secondary primer P2 anneals with HAP and initiates a cycle of the
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polymerization-repairing reaction, which generates vast reporter oligonucleotide R. Then the
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produced generous reporter oligonucleotide binds to the fluorescence-quenched probe, creating a
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stable double-stranded DNA duplex that can be specifically and efficiently cleaved by Endo IV. The
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resulting two DNA fragments, one carrying the fluorophore and the other loading the quencher, are
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both too short to be stably hybridization with reporter oligonucleotide R, which results in the
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activation of the fluorescence signal. At the same time, a new fluorescence-quenched probe anneals
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with the reporter oligonucleotide, thus signal amplification is achieved owing to cyclic annealing and
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cleavage of the fluorescence-quenched probe. In general, the cyclic ERA reaction can not only
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produce numerous copies of the secondary primer for unfolding HAP and triggering
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"extension-excision-cleavage" reaction, but also generate a large number of reporter oligonucleotides
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that are used for efficient fluorescent signal output. Hence, the proposed ERA-based fluorescence
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sensing strategy indeed creates a useful and versatile platform for ultrasensitive and highly specific
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detection of pathogenic bacteria.
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Scheme 1 Illustration of the target-triggered ERA for ultrasensitive fluorescence assay of S.
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Typhimurium.
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3.2 Feasibility characterization of the biosensor
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To verify the feasibility of designed target-aptamer binding triggered ERA strategy for S.
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Typhimurium assay, different fluorescence intensities obtained upon quantifying S. Typhimurium, and
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those obtained in a series of control experiments were depicted in Fig.1. As shown in Fig. 1a, a
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significantly strong peak at around 519 nm was obtained in the presence of S. Typhimurium,
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indicating a substantial number of R combined to fluorescence-quenched probe. In contrast, it was
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found that a negligible peak was achieved for blank sample, suggesting almost no R generated (curve
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b). This clearly revealed that S. Typhimurium triggered ERA reaction with the aid of Bst DNA
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polymerase, UDG and Endo IV, and produced abundant R complementary to fluorescence-quenched
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probe, thus most fluorescence-quenched probe was cleaved by Endo IV leading to enhancing the
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fluorescence intensities. A low peak was observed when S. Typhimurium replaced by non-target
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pathogenic bacteria E. coli (curve c), demonstrating the significantly increased fluorescence signal
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was induced by the specific recognition of S. Typhimurium. And in the absence of HAP, we also
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obtained dinky peak (curve d), which suggesting that HAP can not only act as template for ERA, but
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also be used to generate a great quantity of R sequences for efficient signal output. Thus, this
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multifunctional hairpin is the critical design for this work. For the proof-of-concept experiment, we
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performed other control experiments in differing samples. In the presence of Bst DNA polymerase,
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UDG and Endo IV but dUTP is replaced by dTTP, a tiny peak appeared in the fluorescence
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intensities curves (curve e), which was attributed that it didn’t create AP sites leading to the UDG
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and Endo IV couldn’t work. In the absence of UDG (curve f), Endo IV (curve g) or Bst DNA
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polymerase (curve h) respectively, there are three negligible peaks similar to that of blank sample.
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This signified that the target-aptamer binding trigged ERA was dependent on the combined action of
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Bst DNA polymerase, UDG and Endo IV. On the basis of these results, it was reasonably concluded
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that our designed route should be feasible for S. Typhimurium assay.
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Fig. 1. (A) Fluorescence emission spectra responses of the biosensor obtained upon analyzing
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1.0×105 cfu mL-1 S. Typhimurium(a). Curves b to d are for the control experiments which are
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controlled with no S. Typhimurium target(b); controlled with S. Typhimurium replaced by non-target
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pathogenic bacteria E. coli (c); controlled without HAP(d). (B) Fluorescence emission spectra
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responses of the biosensor obtained upon analyzing 1.0×105 cfu mL-1 S. Typhimurium (a). Curves e to
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h are for the control experiments which are controlled with dUTP replaced by dTTP (e); controlled
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with no UDG (f); controlled with no Endo IV (g); controlled with no Bst DNA polymerase (h).
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3.3 Gel electrophoresis characterization of the ERA-based versatile fluorescence sensing strategy.
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More direct proof of the biosensor mechanism could be acquired through gel electrophoresis
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analysis, as shown in Fig. 2. Lane 1 displayed a bright band of our designed HAP on the image. P1
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incubation with Apt , a new band appeared, indicating the P1 hybridized with Apt to form arched
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probe (Lane 2). After P1 incubation with HAP, a new band with high molecular weight appeared,
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suggesting that P1 can unfold the hairpin structure of HAP(Lane 3). As seen from lane 4, there are
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two bands for positive sample. The top band demonstrated the hybridized P1 with HAP could prime
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the DNA extension in the presence of Bst DNA polymerase, dATP, dGTP, dCTP and dUTP, while the
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band at the bottom indicated the repeated process of cleavage reaction by the combined effect of both
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UDG and Endo IV, replication reaction and strand-displacement reaction could produce numerous
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short ssDNA fragments (P2 and R). In contrast, two wide bands, which are similar with Lane 1 and
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Lane 2, were observed on the lane 5 for blank sample, suggesting that arched probe (P1-Apt duplex)
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can’t unfold the hairpin structure of HAP. In addition, after incubation with P1, HAP, Bst DNA
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polymerase, UDG but without Endo IV, we observed a band similar to the upper band at lane 4
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appeared on the image (lane 6); And P1 incubation with HAP, Bst DNA polymerase, Endo IV but
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without UDG, a band similar to the Lane 6 was observed (Lane7), Lane 6 and Lane 7 are suggesting
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that the target-aptamer binding trigged ERA strategy was implemented owing to the perfect joint
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action by Bst DNA polymerase, UDG and Endo IV, lack of anyone, the work couldn’t be completed.
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Fig. 2. Gel electrophoresis images for ERA reaction. Lane M, DNA marker; Lane 1, HAP; Lane 2,
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P1 and Apt; Lane 3, P1 and HAP; Lane 4, positive sample; Lane 5, blank sample; Lane 6, P1, HAP,
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Bst DNA polymerase and UDG; Lane 7, P1, HAP, Bst DNA polymerase and Endo IV.
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3.4 Optimization of experimental conditions.
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To acquire an optimal analytical performance of sensing strategy based on the target-aptamer
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binding triggered ERA strategy, some experimental conditions including the isothermal ERA reaction
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temperature and time, the concentration of UDG and the concentration of HAP were investigated.
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Typically, temperature of ERA reaction is critical for the efficiency of this sensing strategy. So we
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examined the variance of the (F1 – F0) /F0 value with the reaction temperature, where F1 and F0 were
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the fluorescence emission peak intensity at 519 nm in the presence and absence of S. Typhimurium,
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respectively. As shown in Fig. 3A, the maximum value was obtained at 50 ℃. Thus, we chose 50 ℃
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as the optimal reaction temperature. Moreover, due to the ERA reaction time is also the key of
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efficiency of amplification, we optimized the ERA reaction time in Fig. 3B. It shows the effect of
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different reaction time on the (F1 –F0) /F0 value. It was found that a maximum value was acquired
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after 90 min. Longer reaction time did not obviously change the value. Thereby, 90 min was used as
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the optimal ERA reaction time in the subsequent research. What’s more, the amplifying efficiency
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was greatly depended on the concentration of UDG in that UDG could create more AP site, so we
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optimized the concentration of UDG. Fig. 3C depicted that the maximum value was obtained at the
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concentration of 0.8 U/µL. So, we chose 0.8 U/µL as the optimal UDG concentration. Additionally,
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as the HAP not only act as template for initiating DNA extension, but also be used to generate a great
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quantity of R sequences for efficient signal output, the concentration of HAP is importance for this
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work. In order to ensure the amplifying efficiency, we optimized the concentration of HAP as shown
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in Fig. 3D, the maximum value was obtained at the concentration of 2 µM. Therefore, 2 µM was
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chosen as the optimal HAP concentration in the following experiments.
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Fig. 3. (A) Effect of the ERA reaction temperature on the Fluorescence emission peak of the
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biosensor. (B) Effect of the ERA reaction time of amplification process on the Fluorescence emission
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peak of the biosensor. (C) Effect of the concentration of UDG on the Fluorescence emission peak of
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the biosensor. (D) Effect of the concentration of HAP on the Fluorescence emission peak of the
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biosensor. The concentration of S. Typhimuriumis 1.0×105 cfu mL-1. Error bars are standard
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deviations across three repetitive experiments.
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3.5 Analytical performance of the ERA-based fluorescence sensing strategy for detecting S.
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Typhimurium
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According to the above standard procedures and under the optimal conditions, the sensitivity
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and the dynamic range of the proposed method was investigated towards S. Typhimurium at various
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concentrations. As seen from Fig. 4A, the peak intensity increased with increasing S. Typhimurium
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concentration from 0 to 5.0×106 cfu mL-1. A linear dependence between the peak fluorescence
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intensity and the logarithm of S. Typhimurium concentration was obtained in the range from 10 cfu
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mL-1 to 5.0×106 cfu mL-1, which was over 5 orders of magnitude (Fig. 4B). The linear regression
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equation was F = 44.03 + 144.89×log (CS. Typhimurium / cfu mL-1) with a correlation coefficient of 0.996.
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The limit of detection (LOD) was calculated to be 9.86 cfu mL-1 in terms of the rule of 3 times
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standard deviation over the blank response.
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Fig. 4 (A) Fluorescence emission spectra responses to different concentrations of S. Typhimurium
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(from curve a to m: 0, 10, 50, 1.0×102, 5.0×102, 1.0×103, 5.0×103,1.0×104, 5.0×104,1.0×105,
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5.0×105,1.0×106, 5.0×106 cfu mL-1). (B) The calibration curve of fluorescence intensity for different
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S. Typhimurium concentrations. Error bars are standard deviations across three repetitive 15
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experiments.
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3.6 The selectivity of the biosensor and comparison of previously reported methods The selectivity of our method for S. Typhimurium assay was also examined by the addition of
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three interfering substances including E.coil, Listeria and Bacillus subtilis. As shown in Fig. 5, the
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fluorescence intensities variation in the presence of even 100-fold excess of the non-target bacteria
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were all less than 3%, demonstrating the excellent selectivity towards S. Typhimurium of the
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proposed method.
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Fig. 5 Specificity evaluation of this isothermal Fluorescence assay for S. Typhimurium detection. The
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concentration of S. Typhimurium is 1.0×105 cfu mL-1. The concentration of non-target bacteria is
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1.0×107 cfu mL-1. Error bars are standard deviations across three repetitive experiments.
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Besides, the analytical performance of our method for quantitative assay of S. Typhimurium was
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compared with that of some earlier reported methods. The results are shown in Tab. 2. As compared
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to those reported the present results exhibit ultrasensitivity and low limit of detection. It was found
304
that the sensitivity of our biosensor improved at least by 2 orders of magnitude as compared to the
305
existing methods [29,30,31,32,33,34] for S. Typhimurium assay. What’s more, this homogeneous
306
fluorescence sensing system can be implemented by only one-step that can offer excellent resistance
16
Page 16 of 33
to possible contaminants and this method has the significant advantages of easy to manipulate,
308
time-saving and admirably reproducibility compared with previous reports. As a result, our biosensor
309
might satisfy the requirements for the profiling low abundance of S. Typhimurium due to its very low
310
detection limit and high specificity.
Detection limit (cfu mL-1)
ELISA SPR Fluorescent nanospheres QCM electrochemical chemiluminescence This work
103 - 105 20 – 102 105 – 107 102 - 104 6×102 – 6 ×106 5.0×102 – 5.0 ×105 10-106
103 20 10 102 6×102 1.2×102 9.86
an
[30] [31] [32] [33] [34]
-
te
3.7 Real sample analysis
[29]
d
312 313
Reference
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Detection methods
Detection range (cfu mL-1)
cr
Table 2 Comparison of different assay methods for pathogenic bacteria determination.
M
311
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307
To further validate the universality of this biosensor, the quantitative assay of spiked milk
315
samples was considered. Different concentrations of S. Typhimurium were spiked into the milk
316
samples and measured directly without any pretreatment except for dilution with a buffer solution.
317
The spiked samples were also detected by a classic plate count method and compared with the results
318
obtained by our method. The results are shown in Tab. 3. It was observed that these data obtained
319
using our method was in good agreement with those obtained via the plate count method, and the
320
discrepancies between the two methods were all smaller that 13%. This clearly revealed the proposed
321
approach might hold a great promise for real sample analysis with great accuracy and reliability.
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Table 3 S. Typhimurium analysis in real samples.
322 Spiked
Measured (cfu mL-1)
Recovery (%) ±SD, n=5
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Page 17 of 33
amount Plate count method
This work
Plate count method
1×10
(1.027±0.023)×10
(0.976±0.027)×10
102.7±2.3
97.6±2.7
1×102
(0.984±0.035)×102
(0.968±0.037)×102
98.4±3.5
96.8±3.7
1×103
(1.019±0.014)×103
(1.017±0.025)×103
101.9±1.4
101.7±2.5
1×104
(1.024±0.009)×104
1.037±0.017)×104
102.4±0.9
1×105
(0.984±0.019)×105
(0.994±0.028)×105
98.4±1.9
103.7±1.7
cr
99.4±2.8
us
323
4. Conclusion
an
324
ip t
This work
(cfu mL-1)
In this study, we have developed a novel signal-on fluorescence sensing system for
326
ultrasensitive and highly specific detection of pathogenic bacteria based on target-triggered ERA. An
327
archer probe containing the anti-target aptamer sequence and a primer sequence, which is used for
328
recognizing target and triggering ERA-based "extension-excision-cleavage" reaction. With this
329
signal amplification strategy, the presence of target leads to the formation of numerous reporter
330
oligonucleotide R, which future binds to the fluorescence-quenched probe to creating a stable
331
double-stranded DNA duplex. Then, the double-stranded DNA duplex can be specifically and
332
efficiently cleaved by Endo IV, which results in the activation of the fluorescence signal. Due to this
333
target-aptamer binding triggered ERA stragety, the developed biosensor indeed provides a new
334
paradigm for highly efficient nucleic acid amplification enabling ultrasensitive toward target
335
pathogenic bacteria with detection limits of 9.86 cfu mL-1and a detection range of 5 orders of
336
magnitude, which represents at least 1000-fold improvement compared to the existing methods.
337
Besides, this work can effectively reduce nonspecific background compared with the existing
338
methods. Moreover, on the basis of this homogeneous sensing principle, our biosensing strategy can
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be implemented by only single-step so that simplified operations without the need of sample
340
pretreatment and multiple washing steps. And it offers the advantage of highly specificity, facilitated
341
instrumentation, shortened analysis time. Additionally, the proposed strategy holds the potential of
342
being extended for the detection of aptamer binding molecules and combined with other detection
343
tools such as electrochemistry assay. This biosensor may create a versatile platform in detecting
344
molecules with trace amounts in bioanalysis and molecular diagnosis.
345
Acknowledgements
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This work was supported by Shandong Province Natural Science Funds for Distinguished
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Young Scholars (JQ201410), NSFC (21405060, 1471644), and Shandong Province Natural Science
348
Funds (ZR2015CM027).
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References
350
[1] E. Scallan, P.M. Griffin, F.J. Angulo, R.V. Tauxe, R.M. Hoekstra, Foodborne Illness Acquired in the United
351
States-Unspecified Agents, Emerging Infect. Dis. 17 (2011) 16−22.
352
[2] E. Scallan, R.M. Hoekstra, F.J. Angulo, R.V. Tauxe, M.A. Widdowson, S.L. Roy, J.L. Jones, P.M. Griffin,
353
Foodborne Illness Acquired in the United States—Major Pathogens, Emerging Infect. Dis. 17 (2011) 7−15.
354
[3] A. Jyoti, P. Vajpayee, G. Singh, C.B. Patel, K.C. Gupta, R. Shanker, Identification of Environmental Reservoirs
355
of Nontyphoidal Salmonellosis: Aptamer-Assisted Bioconcentration and Subsequent Detection of Salmonella
356
Typhimurium by Quantitative Polymerase Chain Reaction, Environ. Sci. Technol. 45 (2011) 8996–9002.
357
[4] B.W. Brooks, J. Devenish, C.L. Lutze-Wallace, D. Milnes, R.H. Robertson, S. Berlie, Evaluation of a
358
monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine
359
preputial washing and vaginal mucus samples, J. Vet. Microbiol. 103 (2004) 77−84.
360
[5] A.K. Deisingh, M. Thompson, Detection of infectious and toxigenic bacteria, Analyst 127 (2002) 567−581.
361
[6] Q. Chen, D. Wang, G.Z. Cai, Y.H. Xiong, Y.T. Li, M.H. Wang, H.L. Huo, J.H. Lin, Fast and sensitive detection
362
of foodborne pathogen using electrochemical impedance analysis, urease catalysis and microfluidics, Biosens.
363
Bioelectron. 86 (2016) 770-776.
364
[7] R. Maalouf, C. Fournier-Wirth, J. Coste,; H. Chebib, Y. Saikali, O. Vittori, A. Errachid, J.P. Cloarec, C. Martelet,
365
N. Jaffrezic-Renault, Label-free detection of bacteria by electrochemical impedance spectroscopy: comparison to
366
surface plasmon resonance, Anal. Chem. 79 (2007) 4879-4886.
367
[8] Y.N Guo., Y. Wang, S. Liu, J.H. Yu, H.Z. Wang, Y.L. Wang, J.D. Huang, Label-free and highly sensitive
368
electrochemical detection of E. colibased on rolling circle amplifications coupled peroxidase-mimicking DNAzyme
369
amplification, Biosens. Bioelectron.75 (2016) 315-319.
370
[9] Z. Li, H. Yang, L. Sun, H. Qi, Q. Gao, C. Zhang, Electrogenerated chemiluminescence biosensors for the
Ac ce p
te
d
M
an
us
cr
ip t
349
20
Page 20 of 33
detection of pathogenic bacteria using antimicrobial peptides as capture/signal probes, Sensors and Actuators B:
372
Chemical 210 (2015) 468-474.
373
[10] T.T. Qiu, Y. Wang, J.H. Yu, S. Liu, H.Z. Wang, Y.N. Guo, J.D. Huang, Label-free, homogeneous, and
374
ultrasensitive detection of pathogenic bacteria based on target-triggered isothermally exponential amplification,
375
RSC Adv. 6 (2016) 62031-62037.
376
[11] A. Sahar, A.R.Y.S. Ghadeer, Z. Mohammed, Rapid colorimetric sensing platform for the detection of Listeria
377
monocytogenes foodborne pathogen, Biosens. Bioelectron. 86 (2016) 1061-1066.
378
[12] C. Chen, D.A. Ridzon, A.J. Broomer, Z. Zhou, D.H. Lee, J.T. Nguyen, M. Barbisin, N.L. Xu, V.R. Mahuvakar,
379
M.R. Andersen, K.Q. Lao, K.J. Livak, K.J. Guegler, Real-time quantification of microRNAs by stem–loop
380
RT–PCR, Nucleic Acids Res. 33 (2005) e179.
381
[13] C.L. Manzanares-Palenzuela, I. Mafraa, J. Costaa, M.F. Barrosob, N. de-los-Santos-Álvarezd, C.D. Matosb, M.
382
Beatriz P.P. Oliveiraa, M.J. Lobo-Casta˜ nón, B. López-Ruizc, Electrochemical magnetoassay coupled to PCR as a
383
quantitative approach to detect the soybean transgenic event GTS40-3-2 in foods, Sensors and Actuators B:
384
Chemical 222 (2016) 1050-1057.
385
[14] S. Petralia, R. Verardo, E. Klaric, S. Cavallaro, E. Alessi, C. Schneider, In-Check system: A highly integrated
386
silicon Lab-on-Chip for sample preparation, PCR amplification and microarray detection of nucleic acids directly
387
from biological samples, Sensors and Actuators B: Chemical 187 (2013) 99-105.
388
[15] Y.Q. Cheng, X. Zhang, Z.P. Li, X.X. Jiao, Y.C. Wang, Y.L. Zhang, Highly Sensitive Determination of
389
microRNA Using Target-Primed and Branched Rolling-Circle Amplification, Angew. Chem. Int. Ed. 48 (2009)
390
3268−3272.
391
[16] R. Deng, L. Tang, Q. Tian, Y. Wang, L. Lin, J. Li, Toehold-initiated Rolling Circle Amplification for
392
Visualizing Individual MicroRNAs In Situ in Single Cells, Angew. Chem. Int. Ed. 126 (2014) 2421−2425.
Ac ce p
te
d
M
an
us
cr
ip t
371
21
Page 21 of 33
[17] S.P. Jonstrup, J. Koch, J. Kjems, A microRNA detection system based on padlock probes and rolling circle
394
amplification, RNA 12 (2006) 1747−1752.
395
[18] H.J. Wang, W. Jiang, W. Li, L. Wang, A bicyclo-hairpin probe mediated strand displacement amplification
396
strategy for label-free and sensitive detection of bleomycin, Sensors and Actuators B: Chemical 238 (2017)
397
318-324.
398
[19] C. Shi, Q. Liu, C. Ma, W. Zhong, Exponential strand-displacement amplification for detection of microRNAs,
399
Anal. Chem. 86 (2014) 336−339.
400
[20] H.G. Zhang, F.Y. Li, H.L. Chen, Y.H. Ma, S.D. Qi, X.G. Chen, L. Zhou, AuNPs colorimetric sensor for
401
detecting platelet-derived growth factor-BB based on isothermal target-triggering strand displacement amplification,
402
Sensors and Actuators B: Chemical 207 (2015) 748-755.
403
[21] C.S. Ball, Y.K. Light, C.Y. Koh, S.S. Wheeler, L.L. Coffey, R.J. Meagher, Quenching of Unincorporated
404
Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright,
405
Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses, Anal. Chem. 88 (2016) 3562−3568.
406
[22] J.O. Seung, H.P. Byung, H.J. Jae, C. Goro, D.C. Lee, D.H. Kim, T.S. Seo, Centrifugal loop-mediated
407
isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection, Biosens.
408
Bioelectron. 75 (2016) 293-300.
409
[23] Y. Xia, Z.H. Liu, S.Q. Yan, F. Yin, X.J. Feng, B.F. Liu, Identifying multiple bacterial pathogens by
410
loop-mediated isothermal amplification on a rotate & react slipchip, Sensors and Actuators B: Chemical 228 (2016)
411
491-499.
412
[24] H.M. Choi, V.A. Beck, N.A. Pierce, Next-generation in situ hybridization chain reaction: higher gain, lower
413
cost, greater durability, ACS Nano 8 (2014) 4284-4294.
414
[25] Q. Guo, J.J. Han, S. Shan, D.F. Liu, S.S. Wu, Y.H. Xiong, W.H. Lai, DNA-based hybridization chain reaction
Ac ce p
te
d
M
an
us
cr
ip t
393
22
Page 22 of 33
and biotin–streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA,
416
Biosens. Bioelectron 86 (2016) 990-995.
417
[26] Z.B. Li, X.M. Miao, Z.Y. Cheng, P. Wang, Hybridization chain reaction coupled with the fluorescence
418
quenching of gold nanoparticles for sensitive cancer protein detection, Sensors and Actuators B: Chemical 243
419
(2017) 731-737.
420
[27] X. Liang, K. Jensen, M.D. Frank-Kamenetskii, Very efficient template/primer-independent DNA synthesis by
421
thermophilic DNA polymerase in the presence of a thermophilic restriction endonuclease, Biochemistry 43 (2004)
422
13459−13466.
423
[28] E. Tan, B. Erwin, S. Dames, T. Ferguson, M. Buechel, B. Irvine, K. Voelkerding, A. Niemz, Specific versus
424
nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities,
425
Biochemistry 47 (2008) 9987-9999.
426
[29] E. Nazemi, S. Aithal, W.M. Hassen, E.H. Frost, J.J. Dubowski, GaAs/AlGaAs heterostructure based photonic
427
biosensor for rapid detection of Escherichia coli in phosphate buffered saline solution, Sensors and Actuators B:
428
Chemical 207 (2015) 556-562.
429
[30] E. Bulard, A.B. Spinelli, P. Chaud, A. Roget, R. Calemczuk, S. Fort, T. Livache, Carbohydrates as new probes
430
for the identification of closely related Escherichia coli strains using surface plasmon resonance imaging, Anal.
431
Chem. 87 (2015) 1804-1811.
432
[31] C.Y. Wen, J. Hu, Z.L. Zhang, Z.Q. Tian, G.P. Ou, Y.L. Liao, Y. Li, M. Xie, Z.Y. Sun, D.W. Pang, One-step
433
sensitive detection of Salmonella typhimurium by coupling magnetic capture and fluorescence identification with
434
functional nanospheres, Anal. Chem. 85 (2013) 1223−1230.
435
[32] V.C. Ozalp, G. Bayramoglu, Z. Erdem, M.Y. Arica, Pathogen detection in complex samples by quartz crystal
436
microbalance sensor coupled to aptamer functionalized core-shell type magnetic separation, Anal Chim Acta 853
Ac ce p
te
d
M
an
us
cr
ip t
415
23
Page 23 of 33
(2015) 533-540.
438
[33] X.M. Wang, P. Zhu, F.W. Pi, H. Jiang, J.D. Shao, Y.Z. Zhang, X.L. Sun, A Sensitive and simple
439
macrophage-based electrochemical biosensor for evaluating lipopolysaccharide cytotoxicity of pathogenic bacteria,
440
Biosens. Bioelectron. 81 (2016) 349-357.
441
[34] Z. Li, H. Yang, L. Sun, H. Qi, Q. Gao and C. Zhang, Electrogenerated chemiluminescence biosensors for the
442
detection of pathogenic bacteria using antimicrobial peptides as capture/signal probes, Sensors and Actuators B:
443
Chemical 210 (2015) 468-474.
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Xueqi Leng received her BSc in Environmental Engineering in 2015 from University of Jinan, Jinan,
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China. She is a master course student in University of Jinan. Her current interests are biosensors and
449
optical sensing strategies.
450
Yu Wang received her B.S. in material chemistry from Liaocheng University in 2005 and PhD in
451
analytical chemistry from Hunan University in 2013. Much of the focus of Dr. Wang’s work in the
452
past years has been focused on developing novel biosensing strategies. She is currently a lecturer in
453
the school of biological science and technology at University of Jinan.
454
Su Liu received her BSc in Biotechnology in 2004 and PhD in Environmental Toxicology in 2009
455
from Ocean University of China, Qingdao, China. Now she is serving as an appointed professor in
456
University of Jinan. She has been engaged in the research fields of Environmental Toxicology and
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biological monitoring.
458
Qianqian Pei received her BSc in Biology in 2015 from University of Jinan, Jinan, China. She is a
459
master course student in University of Jinan. Her current interests are biosensors and electrochemistry.
460
Xuejun Cui received her BSc in Biology in 2015 from Tianshan University, Taian, China. She is a
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master course student in University of Jinan. Her current interests are biosensors and optical sensing.
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Yuqin Tu is a bachelor course student in University of Jinan. Her current interests are biosensors and
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optical sensing.
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Xuejiao Liu is a bachelor course student in University of Jinan. Her current interests are biosensors
465
and novel optical sensing strategies.
466
Jiadong Huang received his BSc in Biology in 1996 and MS in Biosensor in 2002 from Shandong
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Normal University, Jinan, China. He received his PhD (in 2006) in Biosensor from Nankai University,
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Tianjin, China. Now he is serving as a specially appointed professor in University of Jinan. He has
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long been engaged in the research fields of biosensors and biological electrochemistry.
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Highlights 1. This novel fluorescence biosensing strategy based on target-triggered enzymatic repairing amplification (ERA) creates a versatile platform for detection of various targets possessing theirs aptamer.
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2. The developed ERA-based biosensing strategy could combat nonspecific background, which is probably attributed that nonspecific extension of nucleotides might incorporate too many lesions to grow into long DNA replicates.
596 597 598 599
3. The specialized design of hairpin probe (HAP) is used as DNA template responsible for producing a great quantity of reporter oligonucleotides and secondary primers, which is crucial for initiating ERA reaction. What’s more, the fluorescence-quenched probe can cyclically be cleaved via Endo IV-catalyzed repairing reaction.
600 601
4. The proposed biosensor exhibits ultrasensitivity toward target pathogenic bacteria with detection limits of 9.86 cfu mL-1and a detection range of 5 orders of magnitude.
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5. To the best of our knowledge, this is the first time that the target-aptamer binding triggered ERA has been utilized for fluorescence sensing for pathogenic bacteria.
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S0925-4005(17)30915-2 http://dx.doi.org/doi:10.1016/j.snb.2017.05.092 SNB 22376
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
11-3-2017 16-5-2017 17-5-2017
Please cite this article as: X. Leng, Y. Wang, S. Liu, Q. Pei, X. Cui, Y. Tu, X. Liu, J. Huang, Enzymatic Repairing Amplification-based Versatile Signal-on Fluorescence Sensing Platform for Detecting Pathogenic Bacteria, Sensors and Actuators B: Chemical (2017), http://dx.doi.org/10.1016/j.snb.2017.05.092 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.
Enzymatic Repairing Amplification-based Versatile Signal-on
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Fluorescence Sensing Platform for Detecting Pathogenic
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Bacteria
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Xueqi Lenga, Yu Wangb, Su Liua*, Qianqian Peic, Xuejun Cuic, Yuqin Tua, Xuejiao Liub, Jiadong
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Huangb,c
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a College of Resources and Environment, University of Jinan, Jinan 250022, P. R. China
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b College of Biological Sciences and Technology, University of Jinan, Jinan 250022, P. R. China
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c Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P. R. China.
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*
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82769122.
Corresponding author. E-mail: [email protected]; Tel.: +86-531-89736122; Fax: +86-531-
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Abstract A novel fluorescence biosensing strategy for ultrasensitive and specific detection of pathogenic
25
bacteria based on target-triggered enzymatic repairing amplification (ERA) has been developed. This
26
strategy relies on target-aptamer binding mediated ERA reaction, which is carried out cyclically with
27
the help of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and
28
endonuclease IV (Endo IV) to produce amplified fluorescence signal. In our assay, the specially
29
designed hairpin probe (HAP) is used as DNA template responsible for producing a great quantity of
30
reporter oligonucleotides and secondary primers, which can initiate a new cycle of
31
polymerization-repairing amplification. Moreover, by the combination of polymerase-catalyzed
32
incorporation of lesion bases with UDG and Endo IV-assisted ERA, multiple cycle of amplification
33
of the recognition event is achieved, enabling ultrasensitive detection of pathogenic bacteria. Under
34
optimal conditions, this biosensor exhibits ultrasensitivity toward target pathogenic bacteria with
35
detection limits of 9.86 cfu mL-1 and a detection range of 5 orders of magnitude. Additionally, the
36
biosensor has the ability of combating nonspecific background. Furthermore, an archer probe
37
containing the anti-target aptamer sequence and a primer sequence is designed, which translates the
38
binding of target to aptamer into the presence of primer sequence, enabling the detection of various
39
targets, such as protein, DNA, small molecular, and any substance possessing its aptamer. Hence, the
40
proposed target-triggered ERA-based signal-on fluorescence sensing strategy indeed create a
41
versatile and useful platform for detection of pathogenic bacteria, related food safety analysis and
42
clinical diagnosis.
43
Keywords: signal-on fluorescence sensing; homogeneous detection; pathogenic bacteria;
44
target-triggered enzymatic repairing amplification
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1. Introduction Foodborne pathogenic bacteria have always been a major threat to human health. In 2011, the
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U.S. Centers for Disease Control and Prevention reported the damage caused by foodborne diseases
48
each year in the United States: they caused 48 million people to fall sick, 128 000 to be hospitalized,
49
and 3000 to die [1,2]. What’s more, the situation in developing countries is even more serious.
50
Besides, the infectious dose of most pathogenic bacteria is as low as 10 colony-forming units (cfu)
51
[3]. Thus, sensitive, robust, low cost, and ready-to-use analysis of pathogenic bacteria represents a
52
critical approach to prevent and control foodborne diseases.
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Currently, the gold standard for bacterial detection remains the culture method. Though great
54
progress in sensitivity and specificity have been made over the years, including the incorporation of
55
chromogenic agars, the culture approach still suffers from time-consuming for routine analysis in the
56
food industry. Culture methods require several days for pre-enrichment, enrichment, selective plating,
57
identification, and confirmation, at which point a contaminated product could have already reached
58
the consumer [4,5]. Thus, the standard culture methods are neither quick nor simple, making their
59
use at the processing plant level cumbersome and limited.
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In recent years, various biosensing technologies, including electrochemical method [6,7,8],
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electrogenerated chemiluminescence assay [9] and colorimetric analysis [10,11], have been
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developed to improve the sensitivity and specificity, reduce cost and shorten analysis time in
63
foodborne pathogenic bacteria analysis. Electrochemical-based assay method possesses the
64
advantages of high sensitivity, but its accuracy and reproductivity are far from ideal.
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Electrogenerated chemiluminescence biosensor has the advantages of high sensitivity and ease of
66
control, but it isn’t a versatile platform for analyst. Colorimetric method offers the ability for visual
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detection, and high portability, however, it hasn’t satisfactory sensitivity. Thereby, it’s still a
68
challenge to develop simple, rapid, low cost and high sensitive pathogenic bacteria assay methods. There are two factors crucial for affecting the performance of the biosensor, namely specificity
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and sensitivity. In order to improve the sensitivity in the detection of pathogenic bacteria, abundant
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nucleic acid amplification technologies is increasingly developed, such as reverse transcription
72
quantitative
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amplification [18,19,20], loop mediated isothermal amplification [21,22,23], hybridization chain
74
reaction [24,25,26]. Most of these amplification reactions involve a key mechanism of
75
“cleavage-replication” cyclical reaction, in which a phosphodiester bond is cleaved cyclically by
76
nicking endonuclease such that 3′ end at the nick site can act as a primer to initiate a new cycle of
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extension reaction with the aid of polymerase. However, it is reported that the combination of
78
nicking endonuclease and DNA polymerase may cause nonspecific amplification even in the absence
79
of DNA template [27, 28], possibly leading to false positive signal in the detection. Thus, it is of
80
great significance to develop alternative mechanisms that is capable of efficient amplification via
81
polymerase with low background.
rolling circle
amplification [15,16,17],
strand-displacement
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Herein, we report the development of a novel fluorescence biosensing strategy for ultrasensitive
83
and highly specific detection of pathogenic bacteria based on target-triggered enzymatic repairing
84
amplification (ERA). The ERA reaction is initiated specifically by target pathogens-aptamer binding
85
and carried out cyclically with the aid of polymerase and two DNA repairing enzymes, uracil-DNA
86
glycosylase (UDG) and endonuclease IV (Endo IV) to produce amplified fluorescence signal. To the
87
best of our knowledge, this is the first time that the target-aptamer binding triggered ERA has been
88
utilized for fluorescence sensing for pathogenic bacteria. Our strategy features with several
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significant aspects. First, a hairpin probe (HAP) is designed to function as DNA template responsible
90
for producing a great quantity of reporter oligonucleotides and secondary primers, which can anneal
91
with another HAP and initiate a new cycle of polymerization-repairing amplification. Moreover, the
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fluorescence-quenched probe can cyclically be cleaved via Endo IV-catalyzed repairing reaction.
93
Thus, multiple cycle of amplification of the recognition event is achieved by the combination of
94
polymerase-catalyzed incorporation of lesion bases with UDG and Endo IV-assisted ERA, enabling
95
ultrasensitive quantity of pathogenic bacteria. Second, the results reveal the developed ERA-based
96
biosensing strategy could combat nonspecific background, which is probably attributed that
97
nonspecific extension of nucleotides might incorporate too many lesions to grow into long DNA
98
replicates. Third, the specialized design of an archer probe containing the anti-target aptamer
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sequence and a universal primer sequence translates the binding of target to aptamer into the
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presence of primer sequence, which enables the detection of various targets, such as protein, DNA,
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small molecular, and any substance possessing its aptamer. Therefore, the target-triggered
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ERA-based signal-on fluorescence sensing strategy indeed creates a versatile and useful platform for
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ultrasensitive and highly specific assay of pathogenic bacteria.
104
2. Materials and methods
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2.1. Materials and reagents
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All oligonucleotides were HPLC-Purified and synthesized by Sangon Biotechnology Co. Ltd.
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(Shanghai, China). The sequences of oligonucleotides are listed in Table 1. E.coli uracil DNA
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glycosylase (UDG), endonuclease IV (Endo IV) and Bst DNA polymerase (large fragment) were
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purchased from New England Biolabs (Ipswich, MA, USA). dATP, dGTP, dCTP, dUTP, and dTTP
110
were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Salmonella Typhimurium
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(KCTC 1925), E. coli (KCTC 2571), Bacillus subtilis (KCTC 1028) and Listeria (KCTC 3569) were
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obtained from Institute of Microbiology Chinese Academy (Beijing, China). Peptone, beef extract
113
powder, bacto-tryptone and bacto-yeast extract were obtained from Qingdao Biological Technology
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Co. Ltd. (Qingdao, China). The agarose and DNA marker were purchased by Takara Biotechnology
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Co. Ltd. (Dalian, China). Other chemicals were of analytical grade and were used without further
116
purification. Ultrapure water obtained from a Millipore filtration system was used throughout.
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Tab. 1 DNA Oligonucleotides Sequence Used in This Work
117
sequence (5’ to 3’) description
Apt
AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA
P1
TCTTTTCCAAAACGGGCATTACT
HAP
GCCTCCGCTTGAGCCCTGTGTCCCGTCATGCGAGTAATGCCCAAA
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Oligonucleotide name
fluorescence-quenched
GCCT(FAM)XCGC(Dabcyl)TTG
d
probe The underlined bases in Apt are matched bases with P1. Three regions in HAP are marked in
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three different colors. The primer annealing region (P1*) is highlighted in blue, the secondary primer
120
annealing region (P2*) is highlighted in red, the reporter coding region (R*) is highlighted in green.
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The fluorescence-quenched probe is with 4-(4-dimethylaminophenyl) diazenylbenzoic acid (Dabcyl)
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quencher and fluorescein isothiocyanate (FAM) fluorophore, X is the tetrahydrofuran abasic site
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mimic.
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2.2 Apparatus
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Gel electrophoresis was conducted using DYCZ-24DN electrophoresis cell (LIUYI, Beijing,
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China) and Bio-Rad Gel imaging system (Bio-Rad, USA). Fluorescence spectra were recorded using
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a Agilent Cary Eclipse spectrofluorometer (Hitachi, Japan) with excitation at 494 nm. The slit was
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set to be 5 nm for both the excitation and the emission. 6
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2.3 Bacterial Strains and Growth Conditions. All bacteria were freshly harvested from the bacterial culture after incubation at 37 °C for 12 h.
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The resulting bacterial samples were centrifuged at 800 rpm for 5 min. The supernatant was removed.
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The bacterial cells were rinsed with PBS solution (10 mM, pH 7.4) through centrifugation at 800 rpm
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for 5 min. The obtained bacterial cells were resuspended in PBS solution (10 mM, pH 7.4). The
134
bacterial number was determined by the bacteria chamber.
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2.4 S. Typhimurium detection procedures
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All samples were prepared in 4 µL Tris-HCl reaction buffer (50mM, pH 7.9 containing 100 mM
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NaCl, 10 mM MgSO2) for the detection of S. Typhimurium. The detailed procedure was as follows:
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Firstly, Apt (3 µL, 10 µM ) and P1 (3 µL, 10 µM) were mixed at 37 ℃ for 30 min and stored at -20
139
ºC until use. Subsequently, HAP (6 µL, 10 µM), fluorescence-quenched probe (3 µL, 20 µM) and
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equal volumes of different concentrations of S. Typhimurium (or other foodborne diseases), Bst DNA
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polymerase (2 µL, 8000 U/mL), UDG (2.4 µL, 10 U/µL), Endo IV (2.4 µL, 20 U/µL), 10 mM dNTPs
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(10 mM dATP, dGTP, dCTP, dUTP each) were mixed at 50 ℃ for 90 min. At last, the above mixed
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solution (30 µL) was mixed with 120 µL of ultrapure water and scanned using Agilent Cary Eclipse
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spectrofluorometer. Unless noted otherwise, all experiments for S. Typhimurium measurements were
145
repeated three times in this study.
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2.5 Gel electrophoresis
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The gel electrophoresis was performed using the DNA samples (5 µL per well) on a 4% agarose
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gel with a fluorescence stain Cyber Gold (0.5 µg/ mL) in 50 mM Tris-borate running buffer (pH 8.2)
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containing 2 mM EDTA at 145 V for 30 min. After electrophoresis, the gel was visualized via
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fluorescence detection using a Bio-Rad Gel imaging system.
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3. Results and Discussion
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3.1 Principle of the ERA-based versatile fluorescence sensing strategy for ultrasensitive detection of
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pathogenic bacteria
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Scheme 1 illustrates the working principle of the fluorescence sensing strategy for ultrasensitive
155
and highly specific assay of pathogenic bacteria based on target-aptamer binding triggered ERA, and
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S. Typhimurium is selected as a model analyte. An arched probe is designed to contain the S.
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Typhimurium aptamer sequence and a primer (P1) sequence, which is complementary to a DNA
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segment (P1*) at the 3’ end of the hairpin probe (HAP). The HAP consists of three regions and two
159
lesion incorporating sites: a primer annealing region (P1*), a secondary primer annealing region
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(P2*), and a reporter coding region (R*) that is used to produce reporter oligonucleotides
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complementary to the fluorescence-quenched probe. The fluorescence-quenched probe is designed to
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have a tetrahydrofuran abasic site mimic flanked in proximity to nucleotides labelled with a
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fluorophore (FAM) and a quencher (Dabcyl), which is complementary to the reporter oligonucleotide
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(R). In the presence of target S. Typhimurium, the arched probe specifically combines to S.
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Typhimurium, which leads to the release of P1. Then P1 hybridizes with HAP and functions as a
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primer to initiate an extension reaction and replicates the DNA template in HAP with the aid of Bst
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DNA polymerase and four nucleotides dATP, dGTP, dCTP and dUTP. It is worth noting that dUTP is
168
used to instead of dTTP to incorporate lesions. So, two uracil (dU) nucleotides are incorporated in
169
the extension product. Immediately, the two lesion sites are excised by UDG-catalyzed hydrolysis
170
reaction, which results in the N-glycosylic bond joining the uracil base to deoxyribose. The produced
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abasic sites (AP site) are subsequently cleaved by Endo IV at the phosphodiester bond upstream to
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the AP site, which generates a secondary primer (P2) and reporter oligonucleotide R. Then, a new
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cycle of "extension-excision-cleavage" reaction occurs with the help of polymerase, UDG, and Endo
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IV, producing a large number of copies of secondary primer P2 and reporter oligonucleotide R.
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Meanwhile, the secondary primer P2 anneals with HAP and initiates a cycle of the
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polymerization-repairing reaction, which generates vast reporter oligonucleotide R. Then the
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produced generous reporter oligonucleotide binds to the fluorescence-quenched probe, creating a
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stable double-stranded DNA duplex that can be specifically and efficiently cleaved by Endo IV. The
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resulting two DNA fragments, one carrying the fluorophore and the other loading the quencher, are
180
both too short to be stably hybridization with reporter oligonucleotide R, which results in the
181
activation of the fluorescence signal. At the same time, a new fluorescence-quenched probe anneals
182
with the reporter oligonucleotide, thus signal amplification is achieved owing to cyclic annealing and
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cleavage of the fluorescence-quenched probe. In general, the cyclic ERA reaction can not only
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produce numerous copies of the secondary primer for unfolding HAP and triggering
185
"extension-excision-cleavage" reaction, but also generate a large number of reporter oligonucleotides
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that are used for efficient fluorescent signal output. Hence, the proposed ERA-based fluorescence
187
sensing strategy indeed creates a useful and versatile platform for ultrasensitive and highly specific
188
detection of pathogenic bacteria.
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Scheme 1 Illustration of the target-triggered ERA for ultrasensitive fluorescence assay of S.
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Typhimurium.
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3.2 Feasibility characterization of the biosensor
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To verify the feasibility of designed target-aptamer binding triggered ERA strategy for S.
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Typhimurium assay, different fluorescence intensities obtained upon quantifying S. Typhimurium, and
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those obtained in a series of control experiments were depicted in Fig.1. As shown in Fig. 1a, a
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significantly strong peak at around 519 nm was obtained in the presence of S. Typhimurium,
197
indicating a substantial number of R combined to fluorescence-quenched probe. In contrast, it was
198
found that a negligible peak was achieved for blank sample, suggesting almost no R generated (curve
199
b). This clearly revealed that S. Typhimurium triggered ERA reaction with the aid of Bst DNA
200
polymerase, UDG and Endo IV, and produced abundant R complementary to fluorescence-quenched
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probe, thus most fluorescence-quenched probe was cleaved by Endo IV leading to enhancing the
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fluorescence intensities. A low peak was observed when S. Typhimurium replaced by non-target
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pathogenic bacteria E. coli (curve c), demonstrating the significantly increased fluorescence signal
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was induced by the specific recognition of S. Typhimurium. And in the absence of HAP, we also
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obtained dinky peak (curve d), which suggesting that HAP can not only act as template for ERA, but
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also be used to generate a great quantity of R sequences for efficient signal output. Thus, this
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multifunctional hairpin is the critical design for this work. For the proof-of-concept experiment, we
208
performed other control experiments in differing samples. In the presence of Bst DNA polymerase,
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UDG and Endo IV but dUTP is replaced by dTTP, a tiny peak appeared in the fluorescence
210
intensities curves (curve e), which was attributed that it didn’t create AP sites leading to the UDG
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and Endo IV couldn’t work. In the absence of UDG (curve f), Endo IV (curve g) or Bst DNA
212
polymerase (curve h) respectively, there are three negligible peaks similar to that of blank sample.
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This signified that the target-aptamer binding trigged ERA was dependent on the combined action of
214
Bst DNA polymerase, UDG and Endo IV. On the basis of these results, it was reasonably concluded
215
that our designed route should be feasible for S. Typhimurium assay.
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Fig. 1. (A) Fluorescence emission spectra responses of the biosensor obtained upon analyzing
218
1.0×105 cfu mL-1 S. Typhimurium(a). Curves b to d are for the control experiments which are
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controlled with no S. Typhimurium target(b); controlled with S. Typhimurium replaced by non-target
220
pathogenic bacteria E. coli (c); controlled without HAP(d). (B) Fluorescence emission spectra
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responses of the biosensor obtained upon analyzing 1.0×105 cfu mL-1 S. Typhimurium (a). Curves e to
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h are for the control experiments which are controlled with dUTP replaced by dTTP (e); controlled
223
with no UDG (f); controlled with no Endo IV (g); controlled with no Bst DNA polymerase (h).
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3.3 Gel electrophoresis characterization of the ERA-based versatile fluorescence sensing strategy.
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More direct proof of the biosensor mechanism could be acquired through gel electrophoresis
226
analysis, as shown in Fig. 2. Lane 1 displayed a bright band of our designed HAP on the image. P1
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incubation with Apt , a new band appeared, indicating the P1 hybridized with Apt to form arched
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probe (Lane 2). After P1 incubation with HAP, a new band with high molecular weight appeared,
229
suggesting that P1 can unfold the hairpin structure of HAP(Lane 3). As seen from lane 4, there are
230
two bands for positive sample. The top band demonstrated the hybridized P1 with HAP could prime
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the DNA extension in the presence of Bst DNA polymerase, dATP, dGTP, dCTP and dUTP, while the
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band at the bottom indicated the repeated process of cleavage reaction by the combined effect of both
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UDG and Endo IV, replication reaction and strand-displacement reaction could produce numerous
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short ssDNA fragments (P2 and R). In contrast, two wide bands, which are similar with Lane 1 and
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Lane 2, were observed on the lane 5 for blank sample, suggesting that arched probe (P1-Apt duplex)
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can’t unfold the hairpin structure of HAP. In addition, after incubation with P1, HAP, Bst DNA
237
polymerase, UDG but without Endo IV, we observed a band similar to the upper band at lane 4
238
appeared on the image (lane 6); And P1 incubation with HAP, Bst DNA polymerase, Endo IV but
239
without UDG, a band similar to the Lane 6 was observed (Lane7), Lane 6 and Lane 7 are suggesting
240
that the target-aptamer binding trigged ERA strategy was implemented owing to the perfect joint
241
action by Bst DNA polymerase, UDG and Endo IV, lack of anyone, the work couldn’t be completed.
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Fig. 2. Gel electrophoresis images for ERA reaction. Lane M, DNA marker; Lane 1, HAP; Lane 2,
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P1 and Apt; Lane 3, P1 and HAP; Lane 4, positive sample; Lane 5, blank sample; Lane 6, P1, HAP,
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Bst DNA polymerase and UDG; Lane 7, P1, HAP, Bst DNA polymerase and Endo IV.
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3.4 Optimization of experimental conditions.
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To acquire an optimal analytical performance of sensing strategy based on the target-aptamer
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binding triggered ERA strategy, some experimental conditions including the isothermal ERA reaction
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temperature and time, the concentration of UDG and the concentration of HAP were investigated.
250
Typically, temperature of ERA reaction is critical for the efficiency of this sensing strategy. So we
251
examined the variance of the (F1 – F0) /F0 value with the reaction temperature, where F1 and F0 were
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the fluorescence emission peak intensity at 519 nm in the presence and absence of S. Typhimurium,
253
respectively. As shown in Fig. 3A, the maximum value was obtained at 50 ℃. Thus, we chose 50 ℃
254
as the optimal reaction temperature. Moreover, due to the ERA reaction time is also the key of
255
efficiency of amplification, we optimized the ERA reaction time in Fig. 3B. It shows the effect of
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different reaction time on the (F1 –F0) /F0 value. It was found that a maximum value was acquired
257
after 90 min. Longer reaction time did not obviously change the value. Thereby, 90 min was used as
258
the optimal ERA reaction time in the subsequent research. What’s more, the amplifying efficiency
259
was greatly depended on the concentration of UDG in that UDG could create more AP site, so we
260
optimized the concentration of UDG. Fig. 3C depicted that the maximum value was obtained at the
261
concentration of 0.8 U/µL. So, we chose 0.8 U/µL as the optimal UDG concentration. Additionally,
262
as the HAP not only act as template for initiating DNA extension, but also be used to generate a great
263
quantity of R sequences for efficient signal output, the concentration of HAP is importance for this
264
work. In order to ensure the amplifying efficiency, we optimized the concentration of HAP as shown
265
in Fig. 3D, the maximum value was obtained at the concentration of 2 µM. Therefore, 2 µM was
266
chosen as the optimal HAP concentration in the following experiments.
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267 268
Fig. 3. (A) Effect of the ERA reaction temperature on the Fluorescence emission peak of the
269
biosensor. (B) Effect of the ERA reaction time of amplification process on the Fluorescence emission
270
peak of the biosensor. (C) Effect of the concentration of UDG on the Fluorescence emission peak of
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the biosensor. (D) Effect of the concentration of HAP on the Fluorescence emission peak of the
272
biosensor. The concentration of S. Typhimuriumis 1.0×105 cfu mL-1. Error bars are standard
273
deviations across three repetitive experiments.
274
3.5 Analytical performance of the ERA-based fluorescence sensing strategy for detecting S.
275
Typhimurium
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According to the above standard procedures and under the optimal conditions, the sensitivity
277
and the dynamic range of the proposed method was investigated towards S. Typhimurium at various
278
concentrations. As seen from Fig. 4A, the peak intensity increased with increasing S. Typhimurium
279
concentration from 0 to 5.0×106 cfu mL-1. A linear dependence between the peak fluorescence
280
intensity and the logarithm of S. Typhimurium concentration was obtained in the range from 10 cfu
281
mL-1 to 5.0×106 cfu mL-1, which was over 5 orders of magnitude (Fig. 4B). The linear regression
282
equation was F = 44.03 + 144.89×log (CS. Typhimurium / cfu mL-1) with a correlation coefficient of 0.996.
283
The limit of detection (LOD) was calculated to be 9.86 cfu mL-1 in terms of the rule of 3 times
284
standard deviation over the blank response.
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285 286
Fig. 4 (A) Fluorescence emission spectra responses to different concentrations of S. Typhimurium
287
(from curve a to m: 0, 10, 50, 1.0×102, 5.0×102, 1.0×103, 5.0×103,1.0×104, 5.0×104,1.0×105,
288
5.0×105,1.0×106, 5.0×106 cfu mL-1). (B) The calibration curve of fluorescence intensity for different
289
S. Typhimurium concentrations. Error bars are standard deviations across three repetitive 15
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290
experiments.
291
3.6 The selectivity of the biosensor and comparison of previously reported methods The selectivity of our method for S. Typhimurium assay was also examined by the addition of
293
three interfering substances including E.coil, Listeria and Bacillus subtilis. As shown in Fig. 5, the
294
fluorescence intensities variation in the presence of even 100-fold excess of the non-target bacteria
295
were all less than 3%, demonstrating the excellent selectivity towards S. Typhimurium of the
296
proposed method.
297
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Fig. 5 Specificity evaluation of this isothermal Fluorescence assay for S. Typhimurium detection. The
299
concentration of S. Typhimurium is 1.0×105 cfu mL-1. The concentration of non-target bacteria is
300
1.0×107 cfu mL-1. Error bars are standard deviations across three repetitive experiments.
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Besides, the analytical performance of our method for quantitative assay of S. Typhimurium was
302
compared with that of some earlier reported methods. The results are shown in Tab. 2. As compared
303
to those reported the present results exhibit ultrasensitivity and low limit of detection. It was found
304
that the sensitivity of our biosensor improved at least by 2 orders of magnitude as compared to the
305
existing methods [29,30,31,32,33,34] for S. Typhimurium assay. What’s more, this homogeneous
306
fluorescence sensing system can be implemented by only one-step that can offer excellent resistance
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to possible contaminants and this method has the significant advantages of easy to manipulate,
308
time-saving and admirably reproducibility compared with previous reports. As a result, our biosensor
309
might satisfy the requirements for the profiling low abundance of S. Typhimurium due to its very low
310
detection limit and high specificity.
Detection limit (cfu mL-1)
ELISA SPR Fluorescent nanospheres QCM electrochemical chemiluminescence This work
103 - 105 20 – 102 105 – 107 102 - 104 6×102 – 6 ×106 5.0×102 – 5.0 ×105 10-106
103 20 10 102 6×102 1.2×102 9.86
an
[30] [31] [32] [33] [34]
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[29]
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312 313
Reference
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Detection methods
Detection range (cfu mL-1)
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Table 2 Comparison of different assay methods for pathogenic bacteria determination.
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To further validate the universality of this biosensor, the quantitative assay of spiked milk
315
samples was considered. Different concentrations of S. Typhimurium were spiked into the milk
316
samples and measured directly without any pretreatment except for dilution with a buffer solution.
317
The spiked samples were also detected by a classic plate count method and compared with the results
318
obtained by our method. The results are shown in Tab. 3. It was observed that these data obtained
319
using our method was in good agreement with those obtained via the plate count method, and the
320
discrepancies between the two methods were all smaller that 13%. This clearly revealed the proposed
321
approach might hold a great promise for real sample analysis with great accuracy and reliability.
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Table 3 S. Typhimurium analysis in real samples.
322 Spiked
Measured (cfu mL-1)
Recovery (%) ±SD, n=5
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amount Plate count method
This work
Plate count method
1×10
(1.027±0.023)×10
(0.976±0.027)×10
102.7±2.3
97.6±2.7
1×102
(0.984±0.035)×102
(0.968±0.037)×102
98.4±3.5
96.8±3.7
1×103
(1.019±0.014)×103
(1.017±0.025)×103
101.9±1.4
101.7±2.5
1×104
(1.024±0.009)×104
1.037±0.017)×104
102.4±0.9
1×105
(0.984±0.019)×105
(0.994±0.028)×105
98.4±1.9
103.7±1.7
cr
99.4±2.8
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323
4. Conclusion
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324
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This work
(cfu mL-1)
In this study, we have developed a novel signal-on fluorescence sensing system for
326
ultrasensitive and highly specific detection of pathogenic bacteria based on target-triggered ERA. An
327
archer probe containing the anti-target aptamer sequence and a primer sequence, which is used for
328
recognizing target and triggering ERA-based "extension-excision-cleavage" reaction. With this
329
signal amplification strategy, the presence of target leads to the formation of numerous reporter
330
oligonucleotide R, which future binds to the fluorescence-quenched probe to creating a stable
331
double-stranded DNA duplex. Then, the double-stranded DNA duplex can be specifically and
332
efficiently cleaved by Endo IV, which results in the activation of the fluorescence signal. Due to this
333
target-aptamer binding triggered ERA stragety, the developed biosensor indeed provides a new
334
paradigm for highly efficient nucleic acid amplification enabling ultrasensitive toward target
335
pathogenic bacteria with detection limits of 9.86 cfu mL-1and a detection range of 5 orders of
336
magnitude, which represents at least 1000-fold improvement compared to the existing methods.
337
Besides, this work can effectively reduce nonspecific background compared with the existing
338
methods. Moreover, on the basis of this homogeneous sensing principle, our biosensing strategy can
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be implemented by only single-step so that simplified operations without the need of sample
340
pretreatment and multiple washing steps. And it offers the advantage of highly specificity, facilitated
341
instrumentation, shortened analysis time. Additionally, the proposed strategy holds the potential of
342
being extended for the detection of aptamer binding molecules and combined with other detection
343
tools such as electrochemistry assay. This biosensor may create a versatile platform in detecting
344
molecules with trace amounts in bioanalysis and molecular diagnosis.
345
Acknowledgements
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This work was supported by Shandong Province Natural Science Funds for Distinguished
347
Young Scholars (JQ201410), NSFC (21405060, 1471644), and Shandong Province Natural Science
348
Funds (ZR2015CM027).
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References
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[1] E. Scallan, P.M. Griffin, F.J. Angulo, R.V. Tauxe, R.M. Hoekstra, Foodborne Illness Acquired in the United
351
States-Unspecified Agents, Emerging Infect. Dis. 17 (2011) 16−22.
352
[2] E. Scallan, R.M. Hoekstra, F.J. Angulo, R.V. Tauxe, M.A. Widdowson, S.L. Roy, J.L. Jones, P.M. Griffin,
353
Foodborne Illness Acquired in the United States—Major Pathogens, Emerging Infect. Dis. 17 (2011) 7−15.
354
[3] A. Jyoti, P. Vajpayee, G. Singh, C.B. Patel, K.C. Gupta, R. Shanker, Identification of Environmental Reservoirs
355
of Nontyphoidal Salmonellosis: Aptamer-Assisted Bioconcentration and Subsequent Detection of Salmonella
356
Typhimurium by Quantitative Polymerase Chain Reaction, Environ. Sci. Technol. 45 (2011) 8996–9002.
357
[4] B.W. Brooks, J. Devenish, C.L. Lutze-Wallace, D. Milnes, R.H. Robertson, S. Berlie, Evaluation of a
358
monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine
359
preputial washing and vaginal mucus samples, J. Vet. Microbiol. 103 (2004) 77−84.
360
[5] A.K. Deisingh, M. Thompson, Detection of infectious and toxigenic bacteria, Analyst 127 (2002) 567−581.
361
[6] Q. Chen, D. Wang, G.Z. Cai, Y.H. Xiong, Y.T. Li, M.H. Wang, H.L. Huo, J.H. Lin, Fast and sensitive detection
362
of foodborne pathogen using electrochemical impedance analysis, urease catalysis and microfluidics, Biosens.
363
Bioelectron. 86 (2016) 770-776.
364
[7] R. Maalouf, C. Fournier-Wirth, J. Coste,; H. Chebib, Y. Saikali, O. Vittori, A. Errachid, J.P. Cloarec, C. Martelet,
365
N. Jaffrezic-Renault, Label-free detection of bacteria by electrochemical impedance spectroscopy: comparison to
366
surface plasmon resonance, Anal. Chem. 79 (2007) 4879-4886.
367
[8] Y.N Guo., Y. Wang, S. Liu, J.H. Yu, H.Z. Wang, Y.L. Wang, J.D. Huang, Label-free and highly sensitive
368
electrochemical detection of E. colibased on rolling circle amplifications coupled peroxidase-mimicking DNAzyme
369
amplification, Biosens. Bioelectron.75 (2016) 315-319.
370
[9] Z. Li, H. Yang, L. Sun, H. Qi, Q. Gao, C. Zhang, Electrogenerated chemiluminescence biosensors for the
Ac ce p
te
d
M
an
us
cr
ip t
349
20
Page 20 of 33
detection of pathogenic bacteria using antimicrobial peptides as capture/signal probes, Sensors and Actuators B:
372
Chemical 210 (2015) 468-474.
373
[10] T.T. Qiu, Y. Wang, J.H. Yu, S. Liu, H.Z. Wang, Y.N. Guo, J.D. Huang, Label-free, homogeneous, and
374
ultrasensitive detection of pathogenic bacteria based on target-triggered isothermally exponential amplification,
375
RSC Adv. 6 (2016) 62031-62037.
376
[11] A. Sahar, A.R.Y.S. Ghadeer, Z. Mohammed, Rapid colorimetric sensing platform for the detection of Listeria
377
monocytogenes foodborne pathogen, Biosens. Bioelectron. 86 (2016) 1061-1066.
378
[12] C. Chen, D.A. Ridzon, A.J. Broomer, Z. Zhou, D.H. Lee, J.T. Nguyen, M. Barbisin, N.L. Xu, V.R. Mahuvakar,
379
M.R. Andersen, K.Q. Lao, K.J. Livak, K.J. Guegler, Real-time quantification of microRNAs by stem–loop
380
RT–PCR, Nucleic Acids Res. 33 (2005) e179.
381
[13] C.L. Manzanares-Palenzuela, I. Mafraa, J. Costaa, M.F. Barrosob, N. de-los-Santos-Álvarezd, C.D. Matosb, M.
382
Beatriz P.P. Oliveiraa, M.J. Lobo-Casta˜ nón, B. López-Ruizc, Electrochemical magnetoassay coupled to PCR as a
383
quantitative approach to detect the soybean transgenic event GTS40-3-2 in foods, Sensors and Actuators B:
384
Chemical 222 (2016) 1050-1057.
385
[14] S. Petralia, R. Verardo, E. Klaric, S. Cavallaro, E. Alessi, C. Schneider, In-Check system: A highly integrated
386
silicon Lab-on-Chip for sample preparation, PCR amplification and microarray detection of nucleic acids directly
387
from biological samples, Sensors and Actuators B: Chemical 187 (2013) 99-105.
388
[15] Y.Q. Cheng, X. Zhang, Z.P. Li, X.X. Jiao, Y.C. Wang, Y.L. Zhang, Highly Sensitive Determination of
389
microRNA Using Target-Primed and Branched Rolling-Circle Amplification, Angew. Chem. Int. Ed. 48 (2009)
390
3268−3272.
391
[16] R. Deng, L. Tang, Q. Tian, Y. Wang, L. Lin, J. Li, Toehold-initiated Rolling Circle Amplification for
392
Visualizing Individual MicroRNAs In Situ in Single Cells, Angew. Chem. Int. Ed. 126 (2014) 2421−2425.
Ac ce p
te
d
M
an
us
cr
ip t
371
21
Page 21 of 33
[17] S.P. Jonstrup, J. Koch, J. Kjems, A microRNA detection system based on padlock probes and rolling circle
394
amplification, RNA 12 (2006) 1747−1752.
395
[18] H.J. Wang, W. Jiang, W. Li, L. Wang, A bicyclo-hairpin probe mediated strand displacement amplification
396
strategy for label-free and sensitive detection of bleomycin, Sensors and Actuators B: Chemical 238 (2017)
397
318-324.
398
[19] C. Shi, Q. Liu, C. Ma, W. Zhong, Exponential strand-displacement amplification for detection of microRNAs,
399
Anal. Chem. 86 (2014) 336−339.
400
[20] H.G. Zhang, F.Y. Li, H.L. Chen, Y.H. Ma, S.D. Qi, X.G. Chen, L. Zhou, AuNPs colorimetric sensor for
401
detecting platelet-derived growth factor-BB based on isothermal target-triggering strand displacement amplification,
402
Sensors and Actuators B: Chemical 207 (2015) 748-755.
403
[21] C.S. Ball, Y.K. Light, C.Y. Koh, S.S. Wheeler, L.L. Coffey, R.J. Meagher, Quenching of Unincorporated
404
Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright,
405
Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses, Anal. Chem. 88 (2016) 3562−3568.
406
[22] J.O. Seung, H.P. Byung, H.J. Jae, C. Goro, D.C. Lee, D.H. Kim, T.S. Seo, Centrifugal loop-mediated
407
isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection, Biosens.
408
Bioelectron. 75 (2016) 293-300.
409
[23] Y. Xia, Z.H. Liu, S.Q. Yan, F. Yin, X.J. Feng, B.F. Liu, Identifying multiple bacterial pathogens by
410
loop-mediated isothermal amplification on a rotate & react slipchip, Sensors and Actuators B: Chemical 228 (2016)
411
491-499.
412
[24] H.M. Choi, V.A. Beck, N.A. Pierce, Next-generation in situ hybridization chain reaction: higher gain, lower
413
cost, greater durability, ACS Nano 8 (2014) 4284-4294.
414
[25] Q. Guo, J.J. Han, S. Shan, D.F. Liu, S.S. Wu, Y.H. Xiong, W.H. Lai, DNA-based hybridization chain reaction
Ac ce p
te
d
M
an
us
cr
ip t
393
22
Page 22 of 33
and biotin–streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA,
416
Biosens. Bioelectron 86 (2016) 990-995.
417
[26] Z.B. Li, X.M. Miao, Z.Y. Cheng, P. Wang, Hybridization chain reaction coupled with the fluorescence
418
quenching of gold nanoparticles for sensitive cancer protein detection, Sensors and Actuators B: Chemical 243
419
(2017) 731-737.
420
[27] X. Liang, K. Jensen, M.D. Frank-Kamenetskii, Very efficient template/primer-independent DNA synthesis by
421
thermophilic DNA polymerase in the presence of a thermophilic restriction endonuclease, Biochemistry 43 (2004)
422
13459−13466.
423
[28] E. Tan, B. Erwin, S. Dames, T. Ferguson, M. Buechel, B. Irvine, K. Voelkerding, A. Niemz, Specific versus
424
nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities,
425
Biochemistry 47 (2008) 9987-9999.
426
[29] E. Nazemi, S. Aithal, W.M. Hassen, E.H. Frost, J.J. Dubowski, GaAs/AlGaAs heterostructure based photonic
427
biosensor for rapid detection of Escherichia coli in phosphate buffered saline solution, Sensors and Actuators B:
428
Chemical 207 (2015) 556-562.
429
[30] E. Bulard, A.B. Spinelli, P. Chaud, A. Roget, R. Calemczuk, S. Fort, T. Livache, Carbohydrates as new probes
430
for the identification of closely related Escherichia coli strains using surface plasmon resonance imaging, Anal.
431
Chem. 87 (2015) 1804-1811.
432
[31] C.Y. Wen, J. Hu, Z.L. Zhang, Z.Q. Tian, G.P. Ou, Y.L. Liao, Y. Li, M. Xie, Z.Y. Sun, D.W. Pang, One-step
433
sensitive detection of Salmonella typhimurium by coupling magnetic capture and fluorescence identification with
434
functional nanospheres, Anal. Chem. 85 (2013) 1223−1230.
435
[32] V.C. Ozalp, G. Bayramoglu, Z. Erdem, M.Y. Arica, Pathogen detection in complex samples by quartz crystal
436
microbalance sensor coupled to aptamer functionalized core-shell type magnetic separation, Anal Chim Acta 853
Ac ce p
te
d
M
an
us
cr
ip t
415
23
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(2015) 533-540.
438
[33] X.M. Wang, P. Zhu, F.W. Pi, H. Jiang, J.D. Shao, Y.Z. Zhang, X.L. Sun, A Sensitive and simple
439
macrophage-based electrochemical biosensor for evaluating lipopolysaccharide cytotoxicity of pathogenic bacteria,
440
Biosens. Bioelectron. 81 (2016) 349-357.
441
[34] Z. Li, H. Yang, L. Sun, H. Qi, Q. Gao and C. Zhang, Electrogenerated chemiluminescence biosensors for the
442
detection of pathogenic bacteria using antimicrobial peptides as capture/signal probes, Sensors and Actuators B:
443
Chemical 210 (2015) 468-474.
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Xueqi Leng received her BSc in Environmental Engineering in 2015 from University of Jinan, Jinan,
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China. She is a master course student in University of Jinan. Her current interests are biosensors and
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optical sensing strategies.
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Yu Wang received her B.S. in material chemistry from Liaocheng University in 2005 and PhD in
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analytical chemistry from Hunan University in 2013. Much of the focus of Dr. Wang’s work in the
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past years has been focused on developing novel biosensing strategies. She is currently a lecturer in
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the school of biological science and technology at University of Jinan.
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Su Liu received her BSc in Biotechnology in 2004 and PhD in Environmental Toxicology in 2009
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from Ocean University of China, Qingdao, China. Now she is serving as an appointed professor in
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University of Jinan. She has been engaged in the research fields of Environmental Toxicology and
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biological monitoring.
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Qianqian Pei received her BSc in Biology in 2015 from University of Jinan, Jinan, China. She is a
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master course student in University of Jinan. Her current interests are biosensors and electrochemistry.
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Xuejun Cui received her BSc in Biology in 2015 from Tianshan University, Taian, China. She is a
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master course student in University of Jinan. Her current interests are biosensors and optical sensing.
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Yuqin Tu is a bachelor course student in University of Jinan. Her current interests are biosensors and
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optical sensing.
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Xuejiao Liu is a bachelor course student in University of Jinan. Her current interests are biosensors
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and novel optical sensing strategies.
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Jiadong Huang received his BSc in Biology in 1996 and MS in Biosensor in 2002 from Shandong
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Normal University, Jinan, China. He received his PhD (in 2006) in Biosensor from Nankai University,
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Tianjin, China. Now he is serving as a specially appointed professor in University of Jinan. He has
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long been engaged in the research fields of biosensors and biological electrochemistry.
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Highlights 1. This novel fluorescence biosensing strategy based on target-triggered enzymatic repairing amplification (ERA) creates a versatile platform for detection of various targets possessing theirs aptamer.
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2. The developed ERA-based biosensing strategy could combat nonspecific background, which is probably attributed that nonspecific extension of nucleotides might incorporate too many lesions to grow into long DNA replicates.
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3. The specialized design of hairpin probe (HAP) is used as DNA template responsible for producing a great quantity of reporter oligonucleotides and secondary primers, which is crucial for initiating ERA reaction. What’s more, the fluorescence-quenched probe can cyclically be cleaved via Endo IV-catalyzed repairing reaction.
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4. The proposed biosensor exhibits ultrasensitivity toward target pathogenic bacteria with detection limits of 9.86 cfu mL-1and a detection range of 5 orders of magnitude.
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5. To the best of our knowledge, this is the first time that the target-aptamer binding triggered ERA has been utilized for fluorescence sensing for pathogenic bacteria.
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