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Rolling chain amplification (RCA) based signal enhanced electrochemical aptasensor for rapid and ultrasensitive detection of ochratoxin A Lin Huang, Jingjing Wu, Lei Zheng, Haishen Qian, Feng Xue, Yucheng Wu, Daodong Pan, Sam B Adeloju, and Wei Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac402228n • Publication Date (Web): 03 Oct 2013 Downloaded from http://pubs.acs.org on October 7, 2013

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Analytical Chemistry

1

Rolling

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electrochemical aptasensor for rapid and ultrasensitive detection of

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ochratoxin A

4

Lin Huang, 1 Jingjing Wu, 1 Lei Zheng, 1 Haishen Qian, 1 Feng Xue,1 Yucheng Wu, 1 Daodong Pan, 3*

5

Samuel B. Adeloju, 1, 2 * Wei Chen 1∗

6 7 8 9 10

chain

amplification

(RCA)

based

signal

enhanced

1. School of Biotechnology & Food Engineering, Anhui Provincial Key Lab of Functional Materials & Devices, Hefei University of Technology, Hefei, 230009, P. R. China 2. School of Applied Sciences & Engineering, Monash University, Churchill, Victoria 3842, Australia 3. Department of Food Science & Technology, Ningbo University, Ningbo, 315211, P. R. China

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Abstract

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A novel electrochemical aptasensor is described for rapid and ultrasensitive

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detection of ochratoxin A based on signal enhancement with rolling circle amplification

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(RCA). The primer for RCA was designed to compose of a two-part sequence, one part

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of the aptamer sequence directed against OTA while the other part was complementary

17

to the capture probe on the electrode surface. In the presence of target OTA, the primer,

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originally hybridized with the RCA padlock, is replaced to combine with OTA. This

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induces the inhibition of RCA and decreases the OTA sensing signal obtained with

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the electrochemical aptasensor. Under the optimized conditions, ultrasensitive detection

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of OTA was achieved with a limit of detection (LOD) of 0.065 ppt (pg/mL), which is

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much lower than previously reported. The electrochemical aptasensor was also

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successfully applied to the determination of OTA in wine samples. This ultrasensitive

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electrochemical aptasensor is of great practical importance in food safety and could be ∗

Corresponding author: [email protected], [email protected] 1

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widely extended to other hazards detections by replacing the sequence of the

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recognition aptamer.

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Keyword: electrochemical aptasensor, rolling circle amplification, signal amplification,

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OTA, food safety

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Introduction

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Ochratoxin A (OTA) is a toxin produced by the Aspergillus ochraceus and

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Penicillium verrucosum which are widely present in various foods, including wheat,

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corn, barley, oats, rye, rice, vegetables, and other crops.1-4 Extensive studies have

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reported that the major violation site of OTA is the liver and kidney of mammalian

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species and it could further cause carcinogenic, hepatotoxic, nephrotoxic and

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immunotoxic effects on most mammalian species.5-6 Considering these potential toxic

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effects of OTA, the International Agency for Research on Cancer (IARC) has classified

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it as a human carcinogen (2B) and the European Commission has established a

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maximum acceptable OTA level at 5 µg/kg for raw cereal grains, 3 µg/kg for all

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cereal-derived products, and 10 µg/kg for soluble coffee (European Commission,

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2006).7 Consequently, the development of reliable and rapid methods for measurement

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of OTA in food samples is of great practical importance for food safety.

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The traditional instrumental methods developed and adopted as the gold standard

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methods for OTA detection include high performance liquid chromatography (HPLC),8-9

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mass

spectrometry

(MS),10

gas

chromatography 2

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(GC)

and

thin

layer

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chromatography(TLC).11 All of these methods are capable of detecting the target OTA

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with high sensitivity, good stability and repeatability. However, when confronted with

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numerous samples and the urgent requirement for on-site and rapid analysis, the

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applications of these instrumental methods are often limited due to the associated high

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cost, time consuming and laborious pretreatments of samples and the need for trained

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personnel. There is, therefore, still an urgent need for the development of suitable

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analytical methods that could permit more rapid and high throughput detection of target

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OTA than possible with existing methods. Since the successful construction of

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monoclonal antibody preparation technique, various rapid immunoassay methods have

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been developed for OTA detection. Enzyme-linked immunosorbent assay (ELISA) and

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related kits have been widely adopted as a powerful tool for on-site measurement and

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screening of OTA. The specific monoclonal antibody against OTA was prepared and

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used for ELISA method to enable its determination in cereal and coffee samples with

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good

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immunochromatographic lateral flow strip has also been developed for rapid and on-site

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detection of OTA.13 Besides the repeated and tedious culture and wash steps of the

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ELISA protocols, the sensing performance of these immunoassay methods, including

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the sensitivity and selectivity, are highly dependent on the quality of the prepared

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antibody. Furthermore, the storage and application conditions of the antibody such as

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the temperature, pH and ionic strength, are strictly defined. All these conditions have

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limited the ability of the existing antibody based methods to meet the current detection

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requirements. Fortunately, the emergence of nucleic acid aptamers has considerable

sensitivity

and

efficiency.12

Subsequently,

3

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a

colloid

gold

labeled

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potential for resolving these issues effectively and it is now been considered as a new

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class of affinity-based probes that rivals antibodies in various analytical fields. The

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specific advantages of aptamers are due to the fact that: (a) they could be easily

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synthesized chemically with high accuracy at a low cost and could be modified with

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various active functional groups; (b) they have much wider target binding range from

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ions and small molecules to macro molecules or even whole cells and, theoretically, any

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target could be screened with specific aptamer using the systematic evolution of ligands

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by exponential enrichment (SELEX); and (c) the application conditions of aptamer are

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much wider than that of antibodies even at high temperature and, especially,

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aptamer-based methods could be repeatedly used just by eluting the sensing interface

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with different pH buffer for recovery. By using an aptamer as the recognition probe,

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various rapid and sensitive detection methods have been developed for detection of

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OTA including colorimetric,14 fluorescent,15-16 lateral flow strip17-18 and electrochemical

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methods.19-21 Furthermore, the use of signal amplification strategies has also attracted

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much attention for realizing ultrasensitive and trace determination of OTA.22-24 Various

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in

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hybridization,25-26 polymerase chain reaction (PCR),27 isothermal strand displacement

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amplification,28 rolling circle amplification (RCA)29-31 and many others, have been

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integrated into the various detection methods to achieve successful ultrasensitive

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detection. We have also previously constructed a folding based electrochemical

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aptasensor for OTA detection.32 However, the use of an electrochemical aptasensor for

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rapid and ultrasensitive detection of OTA with a signal enhancement capability has not

vitro

nucleic

acid

amplification

techniques

4

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including

supersandwich

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been widely reported, except for a study based on RCA with magnetic beads and

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square-wave stripping voltammetry (SWSV).31 Herein, in this research, we utilized an

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OTA specific aptamer as the recognition probe in an electrochemical aptasensor with

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RCA as the signal amplification strategy due to its isothermal and easy operation

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properties. With the signal enhancement strategy, the resulting electrochemical

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aptasensor could detect OTA with an excellent sensitivity and LOD as low as ppf level

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(10-15 g/mL). This represents at least 100-fold improvement when compared with

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traditional methods. This new electrochemical aptasensor could be adopted for on-site

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trace measurement of OTA in food samples and also for quality control of OTA

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contamination.

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2. Experimental section

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2.1. Reagents and materials

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Phi29 DNA polymerase, T4 DNA ligase, ATP and deoxyribonucleoside

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5′-triphosphates mixture (dNTP) were purchased from Sangon (Shanghai, China).

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6-mercapto-l-hexanol (MCH), tris(2-carboxyethyl) phosphine hydrochloride (TCEP)

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and HAuCl4 was purchased from J&K Chemical. OTA was purchased from Beijing

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Century Aoke Ltd. Co. All other reagents were obtained from Sinopharm Chemical

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Reagent Company, China. Various buffer solutions were prepared from analytical grade

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chemicals without further purification and were prepared with ultrapure water (Milli-Q

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A10 system, Millipore, USA). The hybridization buffer used in this study was 10 mM

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Tris buffer (pH 7.4) with 500 mM NaCl and 1 mM MgCl2. Storage buffer used for 5

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oligonucleotides was 10 mM pH 8.0 tris-(hydroxymethyl) aminomethane(Tris-HCl)

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containing1mM ethylenediaminetetraacetic acid (EDTA). The detailed sequence of

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different oligonucleotides probes were as follows: Padlock: 5’phosphate-TGT CTT

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CGC CTG TCC GAT GCT CTT CCT TGA AAC TTC TTC CTT TCT TTC GAC TAA

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GCA CC-3’; Primer: 5’-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG

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ACA GGC GAA GAC AGG TGC TTA GT-3’; Capture DNA: 5’-CCT TGA AAC TTC

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TTC CTT TCT TTC AAA AA-(CH2)6-thiol-3’.

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2.2. Instrumentation

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Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and

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differential pulse voltammetry (DPV) were carried out on a CHI 660D electrochemical

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workstation with a conventional three-electrode system composed of a functionalized

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gold electrode as the working electrode, a platinum wire auxiliary electrode, and a

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saturated calomel electrode (SCE) as the reference electrode. CV measurement was

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performed in 0.1 mol dm−3 KCl solution containing 5.0 mmol dm-3 [Fe(CN)6]3-/4-, the

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potential range was -0.6 to 0.8 V and the scan rate was 100 mV s-1. EIS was also

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performed in 0.1 mol dm−3 KCl solution containing 5.0 mmol dm−3 [Fe(CN)6]3−/4- and

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the frequency range from 1 to 105 Hz at 0.2 V. DPV measurement was carried out in a

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10.0 mL 10 mM pH 7.4 Tris-HCl system and the experiment parameters were as follows:

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initial potential: -0.10 V; final potential: -0.60 V; pulse amplitude: 0.01 V; pulse width:

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0.05 s; sample width:0.0167 s.

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2.3. Surface functionalization of the gold electrode of the electrochemical aptasensor. 6

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Firstly, the gold electrode was modified with capture probe for immobilization of

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the RCA products. Before the modification of capture probe, the electrode was polished

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with 0.05 µm alumina powder to a mirror. After rinsed thoroughly with deionized water,

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the gold electrode was soaked in piranha solution (H2SO4:H2O2, volume ratio 3:1) for 5

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min, followed by a thorough rinse with deionized water and sonicated in ethanol and

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double-distilled water for 5min to remove residual alumina powder, respectively. Then,

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the electrochemical cleaning of the electrode was carried out by scanning between -0.2

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and 1.5 V in H2SO4 (0.5 M) at a scan rate of 100 mV s−1 until a typical stable cyclic

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voltammogram was obtained. Finally, the electrode was rinsed with deionized water and

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dried in a nitrogen environment. 5 µL thiolated capture ssDNA probe (1µM) was

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incubated with 1.5 µL Tris(2-carboxyethyl) phosphine hydrochloride (TECP, 1mM) for

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0.5 h to cleave the disulfide bonds of the probe followed by dropping on the surface of

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pretreated electrode and incubated at 37°C for 2 h. Then, the electrode was rinsed with

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deionized water and washing buffer in sequence. The resulting electrode was then

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immersed into 2 mM MCH solution for 2 h to block the unoccupied site of the electrode

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and eliminate the nonspecific adsorption effect, which is good for the formation of

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well-aligned DNA monolayer. The resulting electrochemical aptasensor was rinsed with

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washing buffer and deionized water, respectively.

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2.4. Rolling circle amplification research.

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5 µL 1 mM primer and 5 µL 1 mM padlock were hybridized in T4 ligase solution

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(40 mM pH 7.5 Tris-HCl, 10 mM MgCl2, and 0.5 mM ATP) at 37 °C for 1 hour and the 7

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final volume of the resulting solution was 20 µL. Then, 5µL OTA solution of various

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concentrations was added and incubated at 37°C for 1 h. After this period, the padlock

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probe was ligated by incubating at 22 °C for 30 min with 10 units of T4 DNA ligase.

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After the ligation, T4 DNA ligase was inactivated at 65 °C for 10 min. The RCA

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reaction was performed by mixing the RCA buffer (40.0 mM pH 7.5Tris-HCl, 50.0 mM

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KCl, 10.0 mM MgCl2 and 5.0 mM (NH4)2SO4), 1 unit of Phi29 DNA polymerase and

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dNTP (500 nM) with abovementioned ligation solution and incubating at 37 °C for 1h.

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Finally, the resulting mixture was immersed in hot water at 65 °C for 10 min to

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inactivate the polymerase.

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2.5. Fabrication of sensing interface of the electrochemical aptasensor.

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The sensing interface of the electrochemical aptasensor was fabricated through

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the hybridization effect between the capture probe and the RCA product. Typically, 10

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µL RCA product was placed onto the surface of electrode and incubated at 37°C for 1 h.

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Of note, the RCA product was heated to 95 °C for 5 min and cooled down to room

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temperature before immobilization on the electrode. Then the electrode was rinsed with

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washing buffer and deionized water, respectively. After hybridization, the electrode was

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immersed in 8×10-5 M MB solution for 10 min. Then, the electrode was rinsed with

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deionized water again and stored in the dark at 4 °C for further use.

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2.6. OTA measurement with the fabricated electrochemical aptasensor.

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Sensing of OTA was carried out with the fabricated electrochemical aptasensor in

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10 mL Tris-HCl buffer (10 mM, pH 7.4) with 500 mM NaCl and 1 mM MgCl2. The 8

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DPV signal of each OTA measurement at different concentrations was recorded with

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applied pulse amplitude of 50 mV and pulse width of 50 ms within a scan range of -0.6

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to 0.1 V. The detection of OTA in red wine samples was carried out directly by diluting

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the wine sample without any further treatment.

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3. Results and discussions

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3.1. Design of electrochemical aptasensor for ultrasensitive detection of OTA.

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Electrochemical detection of OTA is a very conventional and popular analytical

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method. Extensive research has been carried out focused on the signal amplification of

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electrochemical detection methods. In the present study, the signal amplification

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strategy adopted as shown in Figure 1, is the RCA. The primer, containing the sequence

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of the aptamer against OTA, would partially hybridize with the padlock template. Under

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the condition of RCA, the primer could be increased with the repeat sequence of the

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template padlock. Methylene blue (MB) was adopted as the electrochemical redox

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probe based on the specific binding to guanine bases on DNA molecules.33, 34 Once the

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redox probe is attached, the redox signal would also be increased. However, if there was

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any OTA present in the RCA system, OTA would preferentially bind with primer and

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induce the disassociation of primer from the padlock. Under this circumstance, the RCA

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would not be preceded and the primer would not be prolonged. Consequently, the redox

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signal would not be increased. Therefore, with RCA as the signal amplification strategy,

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the redox signal variations were dramatically enhanced even at ultra-low concentrations

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of OTA compared with that of tradition protocols without treatment of signal 9

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amplification. Accordingly, the sensitivity of OTA detection could be greatly improved.

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Evidently, the sensing of OTA could be easily achieved and a relationship could be

200

constructed between the redox signal variations and OTA concentration. Of note, in our

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study, we adopted the capture probe rather than the direct immobilization of primer on

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the surface of electrode. In RCA system, DTT was always used as the antioxidant

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component to maintain the activity of the enzyme. However, DTT is also always used to

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detach the thiol-modified ssDNA probe from the surface of the electrode. This would

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inevitably influence the sensing response in the presence of the target analyte. Under

206

this condition, the sensing results would be not very accurate and with high deviations.

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3.2. Electrochemical characterization of the modification of the electrode

208

(Figure 2)

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The modification of the sensing interface of the electrode was of great importance

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to the achievable magnitude of OTA response. Each step of the sensing interface

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modification was monitored by EIS. In the impedance spectra shown in Figure 2a, the

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diameter of the semicircle is indicative of the magnitude of the electron transfer

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resistance (Ret). Prior to the immobilization of the capture ssDNA probe, the Ret of the

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bare electrode was quite small due to the direct transfer of electron in the sensing

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system. After the immobilization of the capture ssDNA probe on the electrode surface

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by chemisorption, it was obvious that the diameter of the semicircle increased indicating

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an increased Ret value. This increase of the Ret after immobilization of capture probe

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could be attributed to the negative charged phosphate skeleton of the capture ssDNA,

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which could block the electron-transfer of negatively charged [Fe(CN)6]3−/4− from the 10

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electrode surface. Similarly, after the blocking of electrode with MCH and modification

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of RCA product, the Ret was further increased due to the hindrance and repulsive effect

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on electron transfer on the electrode, respectively. The characterization of the electrode

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was also conducted by cyclic voltammetry (CV). As shown in Figure 2b, the redox peak

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current decreased with the modification of the electrode surface with the DNA, blocking

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MCH and RCA in a similar trend to those observed for the EIS measurements. This is

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due to the hindrance of the electron transfer process of the modified DNA probes on the

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electrode which is consistent with the observed increases of the Ret in the EIS

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measurements. Based on the results obtained by both of these electrochemical

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measurements, it is reasonably conclusive that the modification of the electrode and,

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hence, the construction of the sensing interface for OTA was successfully achieved.

231 232

3.3. Characterization of signal amplification of RCA products

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As discussed in the design of the aptasensor, the RCA was carried out in vitro

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rather than in-situ on the electrode. Therefore, before immobilizing the RCA product

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onto the electrode, the products of RCA were analyzed by using agarose gel

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electrophoresis.

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represent the primer, the padlock and the hybridization of primer and padlock,

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respectively. Lane 5 to 8 is the RCA product with different amplification time and it

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could be divided into two categories: (a) after RCA, the primer was prolonged into two

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main products. One migrated into the gel, while the other was too long to migrate and

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was retained in the sample pore; (b) with increasing amplification time, the quantities of

As shown in Figure S1, lanes 2 to 4 of the electrophoresis result

11

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242

both RCA products increased, as illustrated by the much brighter bands in the gel

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(Figure S1). Meanwhile, it was also noted that with the process of RCA the band of

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primer-padlock dsDNA disappeared (lane 8), again indicating the consumption of

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primer into the RCA products. Based on the adopted design principles, the RCA could

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be modulated in the presence of OTA in the samples. We further interrogated this

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research with the agarose gel electrophoresis. Figure 3a clearly demonstrated that there

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are three main products of RCA in the gel (band 1, 2 and 3) and the band brightness of

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these three products decreased with increased concentration in the RCA system.

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Consequently, these observations confirmed the decreased amount of RCA products and

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the effective interference of the RCA with the competitive binding between OTA and

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primers. The semi-quantitative analysis of the RCA products at different OTA

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concentration is also shown in Figure 3b, the peak intensities of the three bands are all

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decreased with the increase OTA in RCA system from sample 1 to 6. Specifically, with

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10 ppb OTA added in RCA system, the peak intensities of three bands in lane 6 are all

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obviously lower than that in lane 1 without OTA in RCA. This phenomenon is

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consistent with the conclusion made above from the gel with naked-eye observation.

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The RCA adopted for signal amplification was also characterized by DPV

259

measurements. From the results shown in Figure 4, it is obvious that the peak current is

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dramatically enhanced after RCA, which is induced by the repeat sequence in the RCA

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product and repeated labeling of MB probes. This protocol was therefore utilized for the

262

detection of OTA.

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(Figure 3)

265

(Figure 4)

266 267

3.4. Optimization of conditions for the signal amplified electrochemical aptasensor

268

Based on the designed signal amplification protocol, the electrochemical

269

aptasensor was developed for ultrasensitive detection of OTA. To ensure optimum

270

performance, some key parameters including the RCA time and the surface density of

271

the capture probe on the electrode were optimized. As the whole research is focused on

272

the signal amplification with RCA, the need to optimize the reaction time of RCA for

273

improvement of the sensitivity of the OTA detection is obvious. Theoretically, the

274

longer the reaction time, the higher the expected peak current for OTA. However, as

275

shown in Figure 5a, in practice the peak current increased at the early stage of the

276

reaction with increasing reaction time up to 60 min, where a maximum peak current was

277

obtained. Beyond a RCA reaction time of 60 min, the DPV response obtained for OTA

278

did not exhibit further increase. The possible reason for this phenomenon could be

279

attributed to the exhaustion of the available substrates for RCA. Meanwhile, with the

280

increase of RCA reaction time, the more RCA products would be generated. Also the

281

saturation of the DPV signal may be induced by the maximum occupancy of the capture

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probe binding sites on the electrode. Consequently, a reaction time of 60 min was

283

adopted as the optimum and employed for all other investigations.

284

Another key factor for improving the sensitivity of the OTA response is the surface

285

density of the capture probe for immobilization of RCA product on the electrode. The 13

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quantitative modification of capture probe was monitored by EIS and CV, respectively.

287

As shown in Figure 5b and c, it can be seen that the modification of the electrode

288

reached saturation at 2 µM of the capture probe. The reason for this saturation could be

289

attributed to the limited surface area of the electrode. Furthermore, the optimal

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concentration of capture probe for immobilization of RCA products is also considered.

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The measurement of RCA immobilization in the presence of different concentration of

292

the capture probe revealed, as shown in Figure 5d, that the DPV signal increased with

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increasing surface density of capture probe on the electrode. The DPV signal reached

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the maximum value only in the presence of 1 µM of capture probe, and further increase

295

reduced the DPV signal. This may be due to the spatial steric effect of the ssDNA at

296

higher concentration and the limited surface area of the electrode, which would hinder

297

the contact and hybridization between capture probe and RCA product. Alternatively, an

298

increase in the surface density of the capture probe on the electrode surface beyond

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certain limit may increase the diffusion barrier and, hence, reduce the magnitude of the

300

OTA response. Therefore, according to the above results, 1 µM of the capture probe was

301

chosen as the optimal concentration for immobilization of capture probe on the

302

electrode.

303

(Figure 5)

304

305 306

3.5. Analytical performance of RCA based signal amplified electrochemical

307

aptasensor 14

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Under the optimized conditions, the fabricated electrochemical aptasensor was

309

adopted for the detection of OTA at different concentrations. As shown in Figure 6a,

310

DPV response for MB decreased gradually with increasing OTA concentration, which is

311

consistent with the competitive replacement principle discussed earlier. As shown in

312

Figure 6b, a linear relationship obtained with OTA concentration from 0.1 ppt to 5 ppb.

313

The calculated limit of detection (3σ) is as low as 0.065 ppt. Compared with the

314

traditional OTA detection methods and aptamer based sensors14, 31, 35, including our

315

previously reported folding-based aptasensor32, the proposed signal amplified

316

electrochemical aptasensor gave an extraordinarily better sensitivity and, hence, much

317

lower limit of detection. A detailed comparison of the achieved limit of detection with

318

those obtained from previous studies is given in Table S1. Furthermore, the achieved

319

linear range with the signal amplified electrochemical aptasensor is quite wide and the

320

dynamic range is extended by more than 10000-fold. Future research attention could

321

focus on the modulation of the dynamic range of the electrochemical aptasensor and

322

also the realization of a controllable sensitivity for different aims.

323

The integration of the selectivity of the fabricated electrochemical aptasensor with

324

the analogues OTA and other toxins were also investigated. As shown in Figure 7, only

325

the target OTA induced an obvious variation of the peak current, while the current

326

variations induced by the analogues or other toxins at the 10-fold concentrations could

327

be ignored. This satisfactory selectivity could be attributed to the good selectivity and

328

high affinity of the aptamer against OTA. These results, therefore, demonstrate that the

15

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selectivity of the fabricated RCA based signal amplified electrochemical aptasensor is

330

suitable for practical application to real samples.

331 332

(Figure 6)

333

(Figure 7)

334 335

On this basis, the analysis of spiked wine samples with the RCA-based

336

electrochemical aptasensor was considered. Different OTA concentrations were spiked

337

into the wine samples and detected directly without any pretreatments except for

338

dilution with a buffer solution. The spiked samples were further quantified by a classic

339

HPLC method and compared with results obtained with the electrochemical aptasensor.

340

From the results in Table 1, it can be seen that the results obtained with the RCA signal

341

amplified electrochemical aptasensor show good correlation with those obtained by

342

HPLC. This further confirms the practical applicability of the developed method for

343

reliable and ultrasensitive OTA detection.

344 345

4. Conclusions

346

A signal amplified electrochemical aptasensor has been successfully developed

347

for ultrasensitive and reliable detection of OTA. The signal amplification technique

348

adopted increased the redox probe loading on the sensing interface of the

349

electrochemical aptasensor and, thus, enhanced the redox signal for OTA detection.

350

More importantly, the RCA was not carried out onto the surface of the electrode directly 16

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and, effectively avoids the interference with the modification process and ensures the

352

stability of the RCA products on the electrode surface. Under optimal conditions, the

353

detection of OTA with this signal amplified electrochemical aptasensor achieved an

354

extraordinary LOD of 0.065 ppt, which is better than those reported for other OTA

355

detection methods. Of much great significance, with the availability of the relevant

356

aptamers, it is possible to extend the signal amplified electrochemical aptasensor to a

357

broader range of targets. This signal amplified electrochemical aptasensor may provide

358

a powerful tool for detection of OTA and other hazards for food safety.

359 360

Acknowledgements:

361

This work is financially supported by the Huangshan Young Scholar Fund of Hefei

362

University of Technology (407-037025), the National Natural Science Foundation of

363

China with grant 31328009, the Science and Technology Research Project of General

364

Administration of Quality Supervision, Inspection and Quarantine of P. R. China

365

(201210127, 201310135), the 12th Five Years Key Programs (2012BAK08B01-2,

366

2012BAK17B10, SS2012AA101001) and the Fundamental Research Funds for the

367

Central Universities (2013HGCH0008, 2012HGCX0003).

368 369

Supporting Information Available

370

This material is available free of charge via the Internet at http://pubs.acs.org.

371 372

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Caption of Figures:

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Figure 1: The schematic diagram of the RCA based signal amplified electrochemical

453

aptasensor for detection of OTA.

454

Figure 2: Characterization of electrode modifications by (a) EIS and (b) CV. Each

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result is for different step of the electrode modification.

456

Figure 3: Agarose gel electrophoresis of OTA induced variation of RCA products at

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different OTA concentrations. (a) Gel image of RCA products, and (b) quantitative

458

analysis of the gel image. Corresponding OTA concentrations for the lanes are: (1) 0, (2)

459

0.001, (3) 0.01, (4) 0.1, (5) 1, and (6) 10 ppb.

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Figure 4: DPV characterization of electrochemical aptasensor with and without signal

461

amplification.

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Figure 5: Optimization of the signal amplified electrochemical aptasensor. (a) Influence

463

of amplification time of RCA; (b) EIS variation with capture probe concentration; (c)

464

CV variation with capture probe concentration; (d) DPV variation with capture probe

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concentration.

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Figure 6: OTA detection with the signal amplified electrochemical aptasensor. (a) DPV

467

signal for OTA detection at different concentrations, and (b) typical calibration curve for 22

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OTA detection.

469

Figure 7: Demonstrated selectivity of the electrochemical aptasensor for the target OTA

470

compared with OTA analogues and other toxins.

471

Table 1: Application of the electrochemical aptasensor to the detection of OTA in wine

472

and comparison with HPLC detection.

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Figure 1

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Figure 1

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Figure 2

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Figure 2

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Figure 3

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Figure 3

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Figure 4

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Figure 4

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Figure 5

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Figure 5

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Figure 6

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Figure 6

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Figure 7

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Figure 7

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582 583

Table 1

584

Spiked Samples

Spiked Amount (ng/mL, ppb)

Measured (ng/mL, ppb)a Aptasensor

Spiked in buffer

HPLC

Recovery (%) (%)±SD, n=5 Aptasensor

HPLC

1

0.5

0.48±0.03

0.51±0.02

96.0±6.4

102±4.1

2

4.5

4.43±0.19

4.52±0.09

98.4±4.2

100.4±2.0

3

2

2.18±0.14

2.05±0.03

109±7.0

102.5±1.5

4

4.5

4.75±0.23

4.58±0.04

105.6±5.1

101.8±0.8

5

0.5

0.54±0.07

0.52±0.03

108.0±9.3

104.3±6.0

6

2

2.16±0.15

2.04±0.02

108.1±7.5

102.3±1.0

Spiked in Red wine

585 586 587 588 589 590 591 592 593 594 31

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595 596 597

TOC graphic

598

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