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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

2

electrochemical aptasensor for rapid and ultrasensitive detection of

3

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

11 12

Abstract

13

A novel electrochemical aptasensor is described for rapid and ultrasensitive

14

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

15

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

16

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,

18

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

19

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

20

the electrochemical aptasensor. Under the optimized conditions, ultrasensitive detection

21

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

22

much lower than previously reported. The electrochemical aptasensor was also

23

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

24

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

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

ACS Paragon Plus Environment


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

26

recognition aptamer.

27 28

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

29

OTA, food safety

30 31

Introduction

32

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

33

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

34

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

35

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

36

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

37

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

38

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

41

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

42

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

43

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

44

The traditional instrumental methods developed and adopted as the gold standard

45

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

46

mass

spectrometry

(MS),10

gas

chromatography 2


(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

51

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

52

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

57

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

58

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

62

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

66

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

68

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

sensitivity

and

efficiency.12

Subsequently,

3


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

76

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

79

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

82

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

83

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

84

in

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

86

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

88

detection. We have also previously constructed a folding based electrochemical

89

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


including

supersandwich

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

92

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

94

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

95

properties. With the signal enhancement strategy, the resulting electrochemical

96

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

97

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

98

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

99

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

100

contamination.

101

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2. Experimental section

103

2.1. Reagents and materials

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Phi29 DNA polymerase, T4 DNA ligase, ATP and deoxyribonucleoside

105

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

106

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

107

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

108

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

109

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

110

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

111

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

112

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



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113

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

114

containing1mM ethylenediaminetetraacetic acid (EDTA). The detailed sequence of

115

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

116

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

117

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

118

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

119

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

120

2.2. Instrumentation

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

washing buffer and deionized water, respectively.

152

2.4. Rolling circle amplification research.

153

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

154

(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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155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

inactivate the polymerase.

164

2.5. Fabrication of sensing interface of the electrochemical aptasensor.

165

The sensing interface of the electrochemical aptasensor was fabricated through

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

2.6. OTA measurement with the fabricated electrochemical aptasensor.

174

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

175

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

177

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

178

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

179

the wine sample without any further treatment.

180 181

3. Results and discussions

182

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

183

Electrochemical detection of OTA is a very conventional and popular analytical

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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



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198

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

199

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

201

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

202

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

203

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

204

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

205

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.

207

3.2. Electrochemical characterization of the modification of the electrode

208

(Figure 2)

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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


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220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231 232

3.3. Characterization of signal amplification of RCA products

233

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

234

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

235

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

236

electrophoresis.

237

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

238

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

239

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

240

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

241

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

243

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

244

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

245

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

246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

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

260

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

261

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

262

detection of OTA.

263 12


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264

(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

282

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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286

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

290

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

291

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

293

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

294

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

299

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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308

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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

329

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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351

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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444

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448

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449

450

451

Caption of Figures:

452

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

455

result is for different step of the electrode modification.

456

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

457

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.

460

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

461

amplification.

462

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

465

concentration.

466

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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468

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.

473

474

475

476

477

478

479

480

481

482

483

484 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

485

486

Figure 1

487

488

489

Figure 1

490

491

492

493

494

495 24


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496

497

Figure 2

498

499

500

Figure 2

501

502

503

504

505

506

507

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

508

509

Figure 3

510

511 512

Figure 3

513

514

515

516

517

518

26


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519

520

Figure 4

521

522 523

Figure 4

524 525 526 527 528 529 530 531 532 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

533 534

Figure 5

535

536 537

Figure 5

538 539 540 541 542 543 544 545 546 28


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547 548

Figure 6

549

550 551 552

Figure 6

553 554 555 556 557 558 559 560 561 562 563 564 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

565 566

Figure 7

567

568 569

Figure 7

570 571 572 573 574 575 576 577 578 579 580 581 30


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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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

595 596 597

TOC graphic

598

32


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