Colorimetric Aptasensor Based on Enzyme for the Detection of Vibrio

Aug 24, 2015 - Sensitive colorimetric immunoassay of Vibrio parahaemolyticus based on specific nonapeptide probe screening from a phage display librar...
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Colorimetric aptasensor based on enzyme for the detection of Vibrio parahaemolyticus Shijia Wu, Yinqiu Wang, Nuo Duan, Haile Ma, and Zhouping Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03224 • Publication Date (Web): 24 Aug 2015 Downloaded from http://pubs.acs.org on August 25, 2015

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Journal of Agricultural and Food Chemistry

Colorimetric aptasensor based on enzyme for the detection of Vibrio parahaemolyticus

Shijia Wu, a Yinqiu Wang, a Nuo Duan,a * Haile Ma, b Zhouping Wang a a

State Key Laboratory of Food Science and Technology, Synergetic Innovation

Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China b

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

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ABSTRACT

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A simple colorimetric aptasensor system has been developed to detect Vibrio

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parahaemolyticus. Magnetic nanoparticles (MNPs) are synthesized and conjugated

4

with specific aptamers against target and used as capture probes. In addition, this

5

method employs gold nanoparticles (AuNPs) as carriers of horseradish peroxidase

6

(HRP) and aptamers, which served as signal probes. In the presence of target, a

7

“sandwich-type” complex of AuNPs-HRP-aptamer-target-aptamer-MNPs is formed

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through specific recognition of aptamers and corresponding target. As a result, HRP

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molecules confine at the surface of the “sandwich” complexes catalyze the enzyme

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substrate, 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, and generate an optical

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signal. Under optimal conditions, the signals are linearly dependent on V.

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parahaemolyticus concentrations from 10 to 106 cfu/mL in a logarithmic plot, with a

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limit of detection 10 cfu/mL. Owning to AuNPs, a large amount of HRP could be

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loaded; resulting in an amplified signal and the sensitivity would be improved. This

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strategy has the potential of being extended to the construction of simple monitor

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systems for a variety of biomolecules related to food safety.

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KEYWORDS: gold nanoparticles, magnetic nanoparticles, Vibrio parahaemolyticus,

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horseradish peroxidase, colometric

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INTRODUCTION

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Vibrio parahaemolyticus, is a gram-negative bacterium, which naturally inhabits

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marine and estuarine environments. 1 It has become one of the most common causes

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of food-borne gastroenteritis, particularly in areas with high seafood consumption.

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Consumption of raw/undercooked seafood results in acute gastroenteritis with the

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following symptoms: headache, abdominal cramps, vomiting, diarrhea and nausea.

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With more and more consumption of seafood, V. parahaemolyticus becomes a food

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safety concern in many Asian countries.

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cases of foodborne illnesses in China. In Chinese province of Guangdong, 29.22%

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outbreaks of food-borne disease were related to V. parahaemolyticus. 5 Therefore, it is

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very important to reduce contamination of seafood with V. parahaemolyticus in order

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to prevent food poisoning and ensure the safety of seafood products.

4

2

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V. parahaemolyticuscauses caused many

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Recently, a variety of analytical methods have been reported for the detection of

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pathogenic bacteria. Polymerase chain reaction (PCR), which is high sensitivity and

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specificity, is a commonly used method for detection of V. parahaemolyticuscauses in

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food and a real-time PCR have the quantitative function compared with the

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conventional PCR. Besides, enzyme-linked immunosorbent assay (ELISA) is the

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most popular and widely used method.

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test a large number of samples at the same time, and could be noticed by naked eyes,

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ELISA has become a powerful tool available for biological research and clinical

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diagnostics. In addition, there are many other immunosensors, which was based on

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antibody−antigen immunoreactions, such as electrochemical methods

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fluorescence methods.

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the quality of the antibodies used. The preparation of the antibodies via animal

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immunization is time-consuming (several months), and the antibodies may become

10,11

6,7

Due to its convenient operation, ability to

8,9

and

However, these immunobioassays are heavily reliant on

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susceptible to stability or modification issues.

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To rival antibodies in these ways, aptamers with high affinity and selectivity are

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beginning to emerge. Aptamers are short single-stranded DNA or RNA molecules

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which were selected through in-vitro selection or the systematic evolution of ligands

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by exponential enrichment (SELEX).

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advantages over antibody.

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synthesis which is higher purity and lower costs. Aptamers can be flexibly modified

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with various chemical tags which could not influence its affinity. Moreover, aptamers

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are small in molecular weight and superior in stability, which can bear repetitious

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denaturation and renaturation. Overall, these unique characteristics make aptamers an

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ideal recognition element for biosensors. As a potential analysis tool in the

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construction of aptasensors, optical analysis has attracted much more interest of

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researchers due to its high sensitivity, quick response and simple operation.

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Nowadays, enzyme linked aptamer assay (ELAA) uses an aptamer as recognition

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element and enzyme as signal readout element has establishing different kinds of

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aptasensors.

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bacteria application.

15,16

14

12,13

Aptamers possess many competitive

Aptamer can be routinely produced by chemical

However, there were seldom reports on ELAA in pathogenic

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Therefore, in this work, we designed an optical strategy for sensitive and specific

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quantitative detection of V. parahaemolyticus by gold nanoparticle-based enzyme-

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linked aptamer sandwich method. MNPs were modified with aptamer to act as the

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capture probe. AuNPs containing a large amout of HRP and aptamer were used as

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signal amplifiers. MNPs-aptamer-target-aptamer-HRP-AuNPs sandwich complexes

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would be formed based on the recognition of aptamers and target. In the addition of

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TMB-H2O2, HRP on the sandwich complexes were catalyzed and generated an optical

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signal. 4

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MATERIALS AND METHODS

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Materials

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Chloroauric acid (HAuCl4), streptavidin and 3,3,5,5-tetramethylbenzidine (TMB)-

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H2O2 were obtained from Sigma-Aldrich (U.S.A.). Horseradish peroxidase (HRP)

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was purchased from Sangon Biotechnology (Shanghai, China). Trisodium citrate, 1,6-

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hexanediamine, anhydrous sodium acetate, FeCl3·6H2O, glycol, 25% glutaraldehyde

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(OHC(CH2)3CHO) and polyethylene glycol (PEG) were of analytical grade and were

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purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The V.

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parahaemolyticus aptamer were prepared in our laboratory

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purified by high-performance liquid chromatography (Sangon Biotechnology, Inc.,

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Shanghai, China). The sequence of V. parahaemolyticus aptamer was 5’-SH-

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TCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGATACT-3’ (apt 1), and

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5’-bio-TCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGATACT-3’ (apt 2).

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and synthesized and

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Instrumentation

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Transmission electron microscopy (TEM) images were acquired with JEM–

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2100HR (TEM, JEOL Ltd., Tokyo, Japan). FT-IR spectra of nanoparticles were

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measured on a Nicolet Nexus 470 Fourier transform infrared spectrophotometer

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(Thermo Electron Co., Boston, U.S.A.). UV-Vis spectra and absorbance were

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obtained using a UV-1800 spectrophotometer (Shimadzu Co., Kyoto, Japan).

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Bacteria strains

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The V. parahaemolyticus ATCC 17802 was kindly donated by the Animal, Plant

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and Food Inspection Centre, Jiangsu Entry-Exit Inspection and Quarantine Bureau

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(Nanjing, China). V. parahaemolyticus was cultured in alkaline peptone with 3% 5

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NaCl (w/v) overnight past the logarithmic phase. One hundred microliters of the

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bacterial culture was diluted with medium and coated on the agar plates and cultured

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at 37 °C for 18 h to count colony forming units. The rest of the bacteria were

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collected carefully by centrifugation at 3000 rpm and 4 °C and washed twice in a

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1×binding buffer (50 mM Tris-HCl at pH 7.4, 5 mM KCl, 100 mM NaCl, and 1 mM

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MgCl2) at room temperature.

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Synthesis of HRP-labeled AuNPs

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Briefly, all glassware used in the experiment were cleaned with aqua regia

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(HNO3/HCl, 3:1, v/v), rinsed thoroughly in ultrapure water, and dried prior to use.

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0.5 mL of 1% HAuCl4 solution and 49.5 mL ultrapure water were heated to boiling

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point for 10 min with continuous stirring. Then, 1.5 mL of 1% trisodium citrate

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was rapidly added, stirred, and boiled for 15 min. The solution colour changed

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from gray to blue, then purple, and finally to wine red during this period. The

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heating source was removed, and the colloid was cooled down to room temperature.

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HRP-labeled AuNPs were prepared according to the previous report, with slight

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modifications.

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with 17.5 µL of HRP molecules (1 mg/mL) to incubate for 10 min at 25 °C under

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gentle shaking. Then, the mixed solution was stood overnight at 4 °C. After incubated

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with PEG (0.5%, w/v) for 30 min at 25 °C, the mixture was centrifuged (12 000 rpm,

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20 min) to remove the unbound HRP molecules and PEG, and washed with PBS for

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three times. The final deposition was suspended in 200 µL of PBS and stored at 4 °C

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for further use.

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The prepared AuNPs (1 mL) was adjusted to pH 8.5 and mixed

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Synthesis of aptamer functionalized HRP-labeled AuNPs 6

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HRP-labeled AuNPs modified by aptamer were prepared according to the literature 19

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with some modification.

This protocol was based on the Au-S interaction between

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the gold lattice and thiolated aptamer. Briefly, 10 µL of 10 µM apt 1 was added to 190

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µL of the already prepared HRP-labeled AuNPs solution and reacted for 16 h. Then

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the HRP-labeled AuNPs-apt 1 complex was aged with salts (0.1 M NaCl, 10 mM

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phosphate, pH 7.0) for 40 h. The prepared complex was centrifuged at 12500 rpm for

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15 min twice to remove the free aptamer. Then, the HRP-labeled AuNPs-apt 1 was

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dispersed in 200 µL of binding buffer for subsequent experiments.

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Synthesis of MNPs and aptamer functionalized MNPs The amine-functionalized MNPs were prepared by a one-step solvothermal method 20

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according to report.

Typically, 6.5 g of 1,6-hexanediamine, 2.0 g of anhydrous

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sodium acetate and 1.0 g of FeCl3•6H2O were dissolved in 30 mL of glycol to form a

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transparent solution with stirring vigorously at 50 °C. The mixture was subsequently

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transferred into a Teflonlined autoclave and heated to 198 °C for 6 h. The product was

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cooled to room temperature and then washed with water and ethanol (2 or 3 times)

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followed by drying at 50 °C.

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The primary amine groups on the surface of MNPs were activated by

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glutaraldehyde, allowing amine groups on aptamer oligonucleotides to be covalently

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attached. Briefly, 1 mg of the MNPs was dispersed in 1 mL of 10 mM phosphate

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buffer solution (PBS, pH 7.4), and 0.25 mL of 25% glutaraldehyde was added into the

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solution. The reaction was continued for 2 h at room temperature gentle shaking,

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followed by washing three times with PBS. The resultant MNPs were dispersed in 1

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mL of 10 mM PBS, and 50 µL of apt 2 (10 µM) were added and incubated for 2 h at

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37 °C with gentle shaking. Next, the MNPs-apt 2 complexes were magnetically 7

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collected and rinsed twice with PBS.

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Procedure for analysis of V. parahaemolyticus

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In a typical experiment, 200 µL of AuNP-HRP-apt 1 and 150 µL of MNPs-apt 2 (1

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mg/mL) were first mixed. Then, 50 µL of binding buffer containing various

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concentrations of V. parahaemolyticus was added and incubated for 30 min at 37 °C

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with gentle shaking. After incubation, the magnetic beads were magnetically collected

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and rinsed with PBS twice. Then, 100 µL of TMB-H2O2 solution was added to each

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tube, and these tubes were incubated at 37 °C for 15 min with gentle shaking. Color

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development was stopped by adding 100 µL of 0.5 M sulfuric acid. After the

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magnetic separation, the absorbance at 450 nm of the supernatant was read with UV

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spectrophotometer.

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Real samples treatment

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The water samples were collected from the Tai Lake, the Yellow sea, laboratory

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and groundwater, China, respectively. Then the water samples were filtered through

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0.45 µm filter. Different V. parahaemolyticus concentrations were then added to the

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prepared samples for the experiments.

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RESULTS AND DISCUSSION

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Principle of the aptasensor

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Scheme 1 shows the principle of this developed aptasensor. MNPs were modified

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with aptamer to act as the capture probe. AuNPs were coated with a large amout of

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HRP and linked to aptamer to act as signal amplifiers. In the presence of target,

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aptamers both on the surface of MNPs and AuNPs bound with the target with high 8

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affinity and specificity, leading to MNPs-aptamer-target-aptamer-HRP-AuNPs

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sandwich complexes formed. With an extra magnetic field, unbound AuNPs-HRP-

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aptamer was removed. In the addition of TMB-H2O2, HRP on the sandwich

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complexes were catalyzed and generated an optical signal. With the carrier of AuNPs,

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a large amount of HRP could be loaded; resulting in an amplified signal and the

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sensitivity would be improved.

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Characteristics of AuNPs and MNPs

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The morphology characteristics of AuNPs were shown in Fig. 1A. The AuNPs are

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monodisperse and spherical with the average size of 15 nm. As shown in Fig. 1B, the

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UV/vis spectrum of AuNPs solution (black line) exhibited a characteristic plasmon

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absorption peak at 520 nm. After modification of HRP on the surface, the UV/vis

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spectrum of the AuNP complex showed a small surface plasmon shift from 520 nm to

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524 nm (red line). The shift after modification of AuNPs with HRP was attributed to

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changes in the particle size and the dielectric nature surrounding the AuNPs due to the

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presence of protein. Both results suggested successful immobilization of HRP onto

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the AuNPs.

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The TEM and FT-IR techniques were used to obtain information about the as-

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synthesized MNPs. Fig. 1C shows TEM image of the MNPs, indicating a good

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dispersibility and morphology. Fig. 1D shows the FT-IR spectra of MNPs. Formation

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of MNPs were confirmed by a strong IR band at 586 cm-1 that comes from the Fe-O

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vibrations. The bands around 1590, 1410 and 1060 cm-1 were NH bending mode and

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C-N stretching vibration, respectively. The results from FT-IR revealed that the

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MNPs have been functionalized with amino groups in the synthetic process.

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Characteristics of the nanoparticles conjugated to aptamer

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We applied UV/vis spectrophotometer to validate the successful coupling of

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thiolated labeled aptamer (apt 1) to AuNPs-HRP through Au-S bonding and biotin

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labeled aptamer (apt 2) to avidin-modified MNPs through biotin-avidin specific

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bonding. As shown in Fig. 2A, AuNPs-HRP shows an absorbance peak at 524 nm

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(curve a), with the addition of apt 1 another peak at 260 nm which is characteristic

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absorption of DNA has been observed (curve b).

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Similarly, no strong absorbance peak was detected for MNPs (curve c). After

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conjugation to avidin, a new absorption peak at approximately 280 nm, which is

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characteristic absorption peak of avidin, was observed (curve d). With the subsequent

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addition of apt 2, the peak at 260 nm was also observed (curve e). These results

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demonstrate that aptamers were successfully coated on the nanoparticles.

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Optimization for the assay

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According to the principle of the assay, experimental parameters such as the

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amount of aptamer functionalized MNPs and aptamer functionalized AuNPs-HRP

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complex would affect the signal response. The effect of the MNPs-apt 2 was first

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studied. As shown in Fig. 3A, Response signals increased with increasing MNPs-apt 2,

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and a maximum was attained at 150 µL (1 mg/mL). Further increase in MNPs-apt 2

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concentration, it had very little additional beneficial effect. Therefore, an optimal

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volume of 150 µL MNPs-apt 2 was chosen in subsequent experiments. Fig. 3B shows

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optimization of the AuNPs-HRP-apt 1 complex concentration. The value of A450 was

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found to increase as the AuNPs-HRP-apt 1 complex concentration was increased until

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200 µL, after which the A450 plateaued and remained constant. Therefore, 200 µL of

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the AuNPs-HRP-apt 1 complex was used for subsequent experiments. 10

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Analytical performance

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Utilizing the optimal conditions in this system, the catalytic ability of the HRP-

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labeled AuNPs on TMB oxidation in the presence of different concentrations of V.

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parahaemolyticus was investigated. Results were evaluated in terms of (A-A0), where

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A0 and A are the absorbance of the colorimetric aptasenosr method in the absence and

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presence of V. parahaemolyticus, respectively. As displayed in Figure 4A, the

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absorbance was increased along with the V. parahaemolyticus concentration. A clear

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change from light yellow to dark yellow could be obviously differentiated by the

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naked eyes (inset A). As shown in Fig. 4B, the A-A0 exhibits a good linear

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relationship with V. parahaemolyticus in the concentration range from 10 to 106

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cfu/mL. The linear regression equation of V. parahaemolyticus is described as

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Y=0.2357x-0.1057 (R2=0.9940), and the detection limit was found to be 10 cfu/mL.

238 239

Specificity, interference and practical performance of the assay

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To investigate the selectivity of the developed method, the AuNPs and MNPs

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complex were employed to detect other pathogenic bacteria, including V.

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alginolyticus, V.vulnificus, V. mimicus, Escherichia coli, Salmonella typhimurium,

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and Staphylococcus aureus at a concentration of 104 cfu/mL, which might affect the

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detection of V. parahaemolyticus in real sample analysis. As shown in Fig. 5, it

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clearly seen that only V. parahaemolyticus induces a dramatic increase of absorbance,

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whereas other species produced signals as low as the blank control. These results

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clearly demonstrated that the developed method is appropriate for the selective

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detection of V. parahaemolyticus.

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In addition, we examined the effect of a variety of possible interfering substances in

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this system. We used a range of proteins, small molecules and ions to determine the 11

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selectivity for V. parahaemolyticus. Under the optimized conditions (the

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concentration of V. parahaemolyticus was set as 104 cfu/mL), none of the other

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interferents affected detection signals, even at concentrations as high as 2% bovine

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serum albumin, 10 µg/mL of immunoglobulin G, and 100 mM of resorcinol,

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microcystic toxins and 500 mM of Na+, K+, Pb2+ and Cd2+. Thus results clearly

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demonstrated that the developed method specifically identified V. parahaemolyticus

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in real environment.

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The practical performance of the developed method was validated with four real

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water samples. Accuracy of this method was evaluated by determining the recoveries

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of V. parahaemolyticus in the water samples by standard addition method. The water

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samples were spiked with V. parahaemolyticus at the desired concentrations (2×102,

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and 2×103 cfu/mL), which was analyzed by standard culture and colony counting

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method. Then, the spiked samples were analyzed by the colorimetric aptasensor

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method. As can be seen from Table 1, the recoveries of the V. parahaemolyticus in the

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four real water samples are between 92.0% and 102.0%. These results show that

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recoveries of V. parahaemolyticus and the reproducibility are satisfactory.

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In summary, we developed a colometric aptasensor to detect V. parahaemolyticus

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in which AuNPs were used as carriers of HRP and aptamer, and MNPs-aptamer were

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used as supporting substrates for capture targets. The AuNPs-HRP-aptamer complex

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loaded a high amount of HRP amplification enzyme, thus the developed assay based

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on the AuNPs complex exhibited improved sensitivity. This feature, as well as its

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convenient magnetic separation, made it a promising alternative to conventional

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pathogenic bacteria methods. This assay system displayed excellent specificity,

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sensitivity, and linearity for quantitative detection of the target molecules, along with

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the production of a color signal that can be detected by the naked eye. In addition, this 12

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method has a potential to detect other bacteria by changing aptamers.

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AUTHOR INFORMATION

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Corresponding Author

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* Phone & Fax: +86-510-85917023; E-mail: [email protected]

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Funding

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This work was partialy supported by NSFC (31401576, 31401575), National

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Science and Technology Support Program of China (2012BAK08B01), JUSRP11547,

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and BK20140155.

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REFERENCE

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(1) Xu, D.; Wang, Y.; Sun, L.; Liu, H.; Li, J. Inhibitory activity of a novel

289

antibacterial

290

parahaemolyticus in shrimp. Food Control. 2013, 30, 58-61.

291

(2) Joseph, S. W.; Colwell, R. R.; Kaper, J. B. Vibrio parahaemolyticus and related

292

halophilic vibrios. Crit Rev Microbiol. 1982, 10, 77-124.

293

(3) Liston, J. Microbial hazards of seafood consumption. Food Technol. 1990, 44, 58-

294

62.

295

(4) Su, Y. C.; Liu, C. Vibrio parahaemolyticus: a concern of seafood safety. Food

296

Microbiol. 2007, 24, 549-558.

297

(5) Zhang, D. S.; Wang, T. Q.; Gu, J. N. Epidemiological analysis on food poisoning

298

in Guangdong 2007-2011. South China J Prev Med. 2013, 39, 74-76.

peptide

AMPNT-6

from

Bacillus

subtilis

13

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against

Vibrio

Journal of Agricultural and Food Chemistry

299

(6) Kumar, B. K.; Raghunath, P.; Devegowda, D.; Deekshit, V. K.; Venugopal, M. N.;

300

Karunasagar, I.; Karunasagar, I. Development of monoclonal antibody based

301

sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in

302

seafood. Int J Food Microbiol. 2011, 145, 244-249.

303

(7) Sakata, J., Kawatsu, K.; Kawahara, R.; Kanki, M.; Iwasaki, T.; Kumeda, Y.;

304

Kodama, H. Production and characterization of a monoclonal antibody against

305

recombinant thermolabile hemolysin and its application to screen for Vibrio

306

parahaemolyticus contamination in raw seafood. Food Control. 2012, 23, 171-176.

307

(8) Zhao, G. Y.; Xing, F. F.; Deng, S. P. A disposable amperometric enzyme

308

immunosensor for rapid detection of Vibrio parahaemolyticus in food based on

309

agarose/Nano-Au membrane and screen-printed electrode. Electrochem Commun.

310

2007, 9, 1263-1268.

311

(9) Sun, W.; Zhang, Y. Y.; Ju, X. M.; Li, G. J.; Gao, H. W.; Sun, Z. F.

312

Electrochemical deoxyribonucleic acid biosensor based on carboxyl functionalized

313

grapheme oxide and ploy-L-lysine modified electrode for the detection of tlh gene

314

sequence related to Vibrio parahaemolyticus. Anal Chim Acta. 2012, 752, 39-44.

315

(10) Wang, L.; Zhang, J. X.; Bai, H. L.; Li, X.; Lv, P. T.; Guo, A. L. Specific

316

detection of Vibrio parahaemolyticus by fluorescence quenching immunoassay based

317

on quantum dots. Appl Biochem Biotech. 2014, 173, 1073-1082.

318

(11) Yi, M. Y.; Ling, L.; Neogi, S. B.; Fan, Y. S.; Tang, D. Y.; Yamasaki, S. J.; Shi,

319

L.; Ye, L. Real time loop-mediated isothermal amplification using a portable

320

fluorescence scanner for rapid and simple detection of Vibrio parahaemolyticus. Food

321

Control. 2014, 41, 91-95.

322

(12) Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment:

323

RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990, 249, 505-510. 14

ACS Paragon Plus Environment

Page 14 of 24

Page 15 of 24

Journal of Agricultural and Food Chemistry

324

(13) Ellington, A. D.; Szostak, J. W. In vitro selection of RNA that bind specific

325

ligands. Nature. 1990, 346, 818-822.

326

(14) Nimjee, S. M.; Rusconi, C. P.; Sullenger, B. A. Aptamers: An emerging class of

327

therapeutics. Annu Rev Med. 2005, 56, 555-583.

328

(15) Nie, J.; Deng, Y.; Deng, Q. P.; Zhang, D. W.; Zhou, Y. L.; Zhang, X. X. A self-

329

assemble aptamer fragment/target complex based high-throughput colorimetric

330

aptasensor using enzyme linked aptamer assay. Talanta. 2013, 106, 309-314.

331

(16) Park, H. Y.; Paeng, I. R. Development of direct competitive enzyme-linked

332

aptamer assay for determination of dopamine in serum. Anal Chim Acta 2011, 685,

333

65-73.

334

(17) Duan, N.; Wu, S. J.; Chen, X. J.; Huang, Y. K.; Wang, Z. P. Selection and

335

identification of a DNA aptamer targeted to Vibrio parahaemolyticus. J. Agr. Food

336

Chem. 2012, 60, 4034-4038.

337

(18) Wu, W. H.; Li, J.; Pan, D.; Li, J.; Song, S. P.; Rong, M. G.; Li, Z. X.; Gao, J. M.;

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Lu, J. X. Gold Nanoparticle-Based Enzyme-Linked Antibody-Aptamer Sandwich

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Assay for Detection of Salmonella Typhimurium. ACS Appl Mater Inter. 2014, 6,

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16974-16981.

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(19) Wang, Y. L.; Wei, H.; Li, B. L.; Ren, W.; Guo, S. J.; Dong, S. J.; Wang, E. K.

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SERS opens a new way in aptasensor for protein recognition with high sensitivity and

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selectivity. Chem Commun. 2007, 48, 5220-5222.

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(20) Wang, L. Y.; Wang, L.; Zhang, F.; Li, Y. D. One-pot synthesis and

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bioapplication

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nanoparticles.

of

amine-functionalized Chem

Eur

magnetite J.

nanoparticles

2006,

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and

hollow

6341-6347.

Journal of Agricultural and Food Chemistry

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Figure captions:

348

Scheme 1. Schematic presentation of V. parahaemolyticus detection using

349

colorimetric aptasensor based on HRP

350

Fig.1 A TEM image of AuNPs (A), the UV/vis spectrum of the AuNPs and HRP

351

modified AuNPs (B), the TEM image (C), and the FT-IR spectrum of MNPs (D)

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Fig. 2 Absorption spectra of bare AuNPs-HRP (a), apt 1-functionalized AuNPs-HRP

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(b), bare MNPs (c), avidin conjugation to MNPs (d), and apt 2-functionalized MNPs

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(e)

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Fig. 3 Optimization of MNPs-apt 2 concentration (A), AuNPs-HRP-apt 1 complex

356

concentration (B). (concentration of V. parahaemolyticus was 104 cfu/mL)

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Fig. 4 (A) Typical recorded output for the detection of different concentrations of

358

bacteria by the developed method. Inset is the color change by the naked eyes, (B)

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Standard curve of the related absorbance (A-A0) versus the concentrations of bacteria.

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Fig. 5 Specificity studies against other bacteria. Concentration of V. parahaemolyticus

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was 104 cfu/mL, while the others was 105 cfu/mL.

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Table 1 Analysis of V. parahaemolyticus cells in the spiked water samples by the

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developed method.

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Table 1 Analysis of V. parahaemolyticus cells in the spiked water samples by the developed method. Sample

Original

Spiked concentration

Measured concentration

Recovery

value(cfu/mL)

(cfu/mL)

(cfu/mL)

(%)

2

2

Tai Lake

0

2.0×10

(1.84±0.15)×10

92.0

seawater

0

2.0×103

(1.97±0.11)×103

98.5

laboratory

0

2.0×102

(1.91±0.28)×102

95.5

groundwater

0

2.0×103

(2.04±0.16)×103

102.0

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Scheme 1.

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B

AuNPs HRP modified AuNPs

1.0

Absorbtion

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0.5

0.0

300

400

500

600

Wavelength (nm)

Fig.1

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Fig. 3

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1.5

////

A

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concentration (cfu mL)

Absorption

6

10 5 10 4 10 3 10 2 10 10 blank

1.0

0.5

0.0 300

400

500

600

700

Wavelength (nm)

1.4

B

1.2 1.0

y=0.2357x-0.1057 2 R =0.9940

A-A0

0.8 0.6 0.4 0.2 0.0 1

2

3

4

5

6

Log Concentration of V. parahaemolyticus (cfu/mL)

Fig. 4

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Fig. 5

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