Screen printed bipolar electrode for sensitive electrochemiluminescence detection of aflatoxin B1 in agricultural products

Journal Pre-proof Screen printed bipolar electrode for sensitive electrochemiluminescence detection of aflatoxin B1 in agricultural products Xiaohui Xiong, Yafei Li, Wei Yuan, Yichen Lu, Xiong Xiong, Yi Li, Xiaoye Chen, Yuanjian Liu PII:

S0956-5663(19)30952-2

DOI:

https://doi.org/10.1016/j.bios.2019.111873

Reference:

BIOS 111873

To appear in:

Biosensors and Bioelectronics

Received Date: 25 September 2019 Revised Date:

10 November 2019

Accepted Date: 11 November 2019

Please cite this article as: Xiong, X., Li, Y., Yuan, W., Lu, Y., Xiong, X., Li, Y., Chen, X., Liu, Y., Screen printed bipolar electrode for sensitive electrochemiluminescence detection of aflatoxin B1 in agricultural products, Biosensors and Bioelectronics (2019), doi: https://doi.org/10.1016/j.bios.2019.111873. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

1

Screen printed bipolar electrode for sensitive electrochemiluminescence detection

2

of Aflatoxin B1 in agricultural products

3

Xiaohui Xionga, Yafei Lia, Wei Yuana, Yichen Lua, Xiong Xionga, Yi Lia, Xiaoye

4

Chena,*, Yuanjian Liua,*

5 6

a

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*Corresponding author. Tel.: 86-25-58139432; Fax: 86-25-58139527

8

E-mail address: [email protected]; [email protected]

9

Coll Food Sci & Light Ind, Nanjing Tech University, Nanjing 211816, China

10

Abstract:

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In order to avoid the occurrence of false positives and false negatives caused by

12

improper pretreatment during the detection of aflatoxin B1 by enzyme linked

13

immunosorbent assay (ELISA). In this paper, we developed a screen printed bipolar

14

electrode (BPE) for sensitive electrochemiluminescence (ECL) detection of aflatoxin

15

B1 in agricultural products. The sensor uses a cathode of closed BPE as a functional

16

sensing interface and an anode as a signal collection interface. In this way, the analyte

17

does not need to participate in the ECL reaction of the anode. It avoids direct contact

18

of photoactive molecules with complex reaction systems and greatly broadens the

19

range of applications for ECL. After mixing the test sample with a known fixed

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concentration of horseradish peroxidase-labeled AFB1 (HRP-AFB1), they compete

21

for binding to monoclonal antibodies. HRP catalyzes the polymerization of aniline to

22

form polyaniline (PANI). Thereby causing a change in the oxidation-reduction

23

potential and the ECL intensity in the electrochemical system, and then achieve the

24

purpose of detecting the AFB1 concentration in the sample. As a result, the sensor has

25

a good analytical performance for AFB1 with a linear range of 0.1-100 ng mL-1 and a

26

detection limit of 0.033 ng mL-1. The sensor avoids the direct contact between the

27

reaction system and the signal measurement system. In recovery experiment for six

28

grains, the results demonstrate that the recovery rate and accuracy of this sensor is

29

better than that of ELISA. This method provides a new idea for the detection of other

30

mycotoxins in grains.

31

Keywords: ECL, BPE, AFB1, PANI, ELISA, grain

32

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1. Introduction

34

Mycotoxins are secondary metabolites produced during the growth and

35

reproduction of fungi. It is usually not destroyed by food grain processing or food

36

cooking heating. At the same time, mycotoxins have various structures, high toxicity

37

and high chemical stability (Turner et al., 2015). Among them, aflatoxin B1 is the

38

most carcinogenic one. Aflatoxin B1 is highly toxic to humans and animals, and its

39

toxic effect is mainly damage to the liver (Wang et al., 2016). Traditional methods for

40

detecting mycotoxins include thin layer chromatography (TLC) (Sana et al., 2019),

41

high performance liquid chromatography (HPLC) (Munawar et al., 2019), gas

42

chromatography (GC) (Ji et al., 2019) and enzyme-linked immunosorbent assay

43

(ELISA) (Sompunga et al., 2019). Although these methods can accurately measure

44

mycotoxins, they require skilled operators, complex pre-treatments, and expensive

45

instruments. Moreover, there is a lack of accuracy in low concentration analysis. More

46

importantly, these methods are extremely susceptible to false positives due to

47

improper pretreatment (Kolosova et al., 2006; Jiang et al., 2013). Therefore, it is

48

necessary to develop a sensitive, rapid and specific analytical technique for detecting

49

mycotoxins to avoid the appearance of false positives.

50

Electrochemiluminescence (ECL) is a combination of chemiluminescence and

51

electrochemistry (Liu et al., 2015). By applying a certain voltage on the electrode,

52

electron transfer between the electrical biomass or the electrical biomass and other

53

components in the system forms an excited state. Luminescence occurs when the

54

excited state returns to the ground state (Wu et al., 2014; Zhao et al., 2015).

55

Compared with the traditional photoluminescence analysis method, the ECL method

56

does not need to excite the light source. It is not affected by the luminescent

57

impurities and scattered light. At the same time, the ECL sensing system has almost

58

no background noise, and the sensitivity and signal-to-noise ratio are significantly

59

improved (Li et al., 2019). The high sensitivity of ECL has made it widely used in

60

biosensing and immunoassay. It has become one of the main research methods in the

61

field of life analysis chemistry (Shi et al., 2016; Zhang et al., 2016; Feng et al., 2015;

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Zhang et al., 2013; Guo et al., 2014; Chow et al., 2009; Wei et al., 2019; Wei et al.,

63

2012). Lv et al. describe a multi-system driven ECL biosensor that utilizes

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competitive catalysis and steric hindrance effects by assembling hemin/G-quadruplex

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on carbon nitride nanosheets (Lv et al., 2018). The integrated dynamic range of the

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detectable concentration for each mechanism is achieved in a single sensor interface.

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Xing’s group constructed a sandwich quenching ECL immunosensor for insulin

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detection, which has a wide detection range and low detection limit (Xing et al.,

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2018).

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Bipolar electrode (BPE) is formed by an electron conductor immersed in an

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ion-conducting phase (Zhang et al., 2019). It usually placed in the microchannel of the

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solution. When a voltage is applied across the microchannel, the difference in

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potential between the solution and the BPE is such that one end is an anode and the

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other end is a cathode (Wang et al., 2018). If the voltage reaches a critical value, then

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the Faraday reaction occurs simultaneously at both ends of the BPE. In addition, BPE

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can physically isolate the reaction system from the signal measurement system for

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miniaturization and integration (Guerrette et al., 2013; Hotta et al., 2002; Plana et al.,

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2010; Wu et al., 2014; Zhan et al., 2002). As an emerging technology, BPE-ECL

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technology has a great advantages in bioanalysis. It not only avoids the direct contact

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of photoactive molecules with complex reaction systems, but also increases the

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amount of information obtained in a single analysis. Moreover, the separate

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modification of the cathode and anode enables highly sensitive ECL detection (Wang

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et al., 2018). Xu’s group used BPE-ECL technology to detect tumor markers such as

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ATP, PSA and AFP (Wu et al., 2015). Khoshfetrat’s group make use of the principle of

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a closed bipolar electrode, the aptamer of aflatoxin M1(AFM1) is modified at the

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cathode of the BPE to achieve quantitative detection of AFM1 in milk (Khoshfetrat et

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al., 2018).

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In this article, we provide a sensitive BPE-ECL mechanism for the determination

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of aflatoxin B1 in agricultural products. It can physically isolate the reaction system

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from the signal measurement system. Avoid the occurrence of false positives and false

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negatives due to improper pretreatment. As illustrated in Scheme 1, a screen printed

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BPE is prepared, and gold nanoparticles (AuNPs) are introduced by gold plating at the

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cathode of BPE. The SH-PEG-COOH is immobilized on the surface of AuNPs by a

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gold-sulfur bond. AFB1 antibody was assembled on the cathode via EDC/NHS

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coupling method. At this point, the functional sensing interface is build. Then, 100 ng

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mL-1 HRP-AFB1 and different concentrations of the test sample will compete with the

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monoclonal antibody on the functional sensing interface. Part of HRP-AFB1 was

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assembled on the cathode based on the antigen-antibody reaction. HRP will catalyze

99

the polymerization of aniline to form polyaniline, which will cause the change of ECL

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and luminescence voltage of the anode of BPE. Finally, the signal is collected and

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analyzed by MPI-E to realize the detection of AFB1. A detailed investigation of

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sensing principle of screen printed BPE is illustrated in Fig. S1.

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Scheme 1. Schematic diagram of AFB1 detection in agricultural products based on

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screen printed BPE-ECL biosensor.

106 107

2. Experiment section

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2.1. Chemicals and reagents

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Aflatoxin B1 murine mAb (AFB1 mAb), horseradish peroxidase-labeled AFB1

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(HRP-AFB1), Aflatoxin B1-BSA (AFB1-BSA) were purchased from Biological

111

Technology Co., Ltd (Shanghai, China). Aflatoxin M1 (AFM1), zearalenone (ZEN),

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ochratoxin A (OTA), deoxynivalenol (DON), and patulin were obtained from Romer

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Labs co., Ltd. (Washington, USA) Chloroauric acid trihydrate (HAuCl4·3H2O),

114

terpyridine ruthenium chloride hexahydrate (Ru(bpy)3Cl2·6H2O), tri-n-propylamine

115

(TPA),

116

2-mercaptoethyl ether acetic acid (SH-PEG-COOH, catalog number: 757810-500MG,

117

average

118

N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich Co. Ltd. (St.

119

Louis, MO). DNA (PolyA59) was purchased from Sangon Biotech Co. Ltd. (Shanghai,

120

China). All other reagents were of analytical grade and used as received.

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aniline,

Mn

hydrogen

1,000),

peroxide

(30%

H2O2),

poly(ethylene

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

glycol)

(EDC),

The buffer solutions employed in this study were as follows: PBS (pH 7.2 ~ 7.4,

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136.89 mM NaCl, 2.67 mM KCl, 8.24 mM Na2HPO4, 1.76 mM NaH2PO4);

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Electrodepositing AuNPs buffer (PBS, 1% HAuCl4·3H2O); Polyaniline (PANI)

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deposition buffer (100 mM acetic acid-sodium acetate (HAc-NaAc), pH 4.3, 200 mM

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aniline, 20 mM H2O2, 0.5 µM polyA59, prepared daily); ECL detection solution (PBS,

126

10 mM Ru(bpy)32+, 50 mM TPA, prepared daily). All solutions were prepared using

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ultrapure water (18.2 MΩ cm at 25 °C) from a Pure Water system (GWA-UN1-F40,

128

Persee, China).

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2.2. Apparatus

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Electrochemical analyzer (CHI 750E) (Chenhua, Shanghai, China), scanning

131

electron microscopy (SEM, S-4800, Hitachi, Japan), MPI-E ECL system (Xi'an

132

Remex Electronics Co. Ltd., Xi’ an, China).

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2.3. Preparation of screen printed bipolar electrode

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The screen printed BPE is prepared as shown in Fig. S2. Firstly, polyethylene

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terephthalate (PET) cheap electrical inert material is used as the substrate. Secondly,

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print two working electrode leads with silver ink at both ends of the substrate. Thirdly,

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drying the substrate, carbon electrode is printed with carbon paste slurry between two

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working electrodes and dried as a BPE. Then, the electrode specification layer is

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printed with a photo-solid insulating paste and cured by ultraviolet light. Finally, the

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electrode insulation layer is printed with a light-solid insulating paste and cured by

141

ultraviolet light. The prepared screen-printed BPE is 3 cm long and 1 cm wide, and

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the length of the bipolar electrode wire is 12 mm.

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2.4. Functional sensing interface construction

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First, 20 µL PBS containing 1% HAuCl4·3H2O was added to the cathode of BPE.

145

20 µL PBS was added to the anode. A certain scanning voltage (3.0 V - 6.0 V) was

146

applied to both ends of the screen printed BPE. As HAuCl4·3H2O gets electrons at the

147

cathode of BPE, the cathode gradually turns yellow. It indicated that gold

148

nanoparticles (AuNPs) are deposited to the cathode. The as-prepared gold-plated BPE

149

was cleaned with ultrapure water and dried in air. Then the cathode of the gold-plated

150

BPE was immersed in 1 mM SH-PEG-COOH solution and incubated for 8 h at room

151

temperature. Then, the cathode was immersed in a mixed solution of EDC and NHS

152

and incubated for 30 min at room temperature. Finally, the AFB1 mAb was added to

153

the cathode and incubated for 2 h at room temperature in the dark. At this point, the

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functional sensing interface is build.

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2.5. Measurement procedure

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20 µL of different concentrations of the test sample and 100 ng mL-1 HRP-AFB1

157

were mixed. Then, the mixture solution was dropped on the functional sensing

158

interface for 2 h incubation. Part of HRP-AFB1 was assembled on the cathode based

159

on the antigen-antibody reaction. The functional sensing interface was then washed

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twice with PBS. Then, 20 µL PANI deposition buffer was added to the functional

161

sensing interface and incubated for 2 h at room temperature in the dark. Finally, 20 µL

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ECL detection solution was added to signal collection interface and the ECL

163

experiments were performed directly without cleaning the functional sensing interface.

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The ECL-voltage curves were obtained by applying a linearly increasing voltage (0 V

165

- 5 V) on the two ends of BPE with the scan rate of 0.1 V s-1. PMT was set at 200 V in

166

the process of detection.

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Fig. 1. Representative SEM images of bare BPE (A), the AuNPs deposited BPE

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obtained after 10 (B), 20 (C), and 30 (D) scanning cycles of cyclic voltammetry.

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Cathode: 20 µL PBS containing 1% HAuCl4·3H2O; Anode: 20 µL PBS; Scanning

171

voltage (3.0 V - 6.0 V); Scan rate: 0.1 V s-1. The inset shows amplified SEM images

172

of cathode of BPE. (E) ECL performance of Ru(bpy)32+ on bare BPE (a) and AuNPs

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deposited BPE obtained after 10 (b), 20 (c), and 30 (d) scanning cycles of cyclic

174

voltammetry in PBS that containing 10 mM Ru(bpy)32+, 50 mM TPA, prepared daily.

175

PMT was set at 200 V. The inset optical images were AuNPs deposited BPE (up) and

176

bare BPE (down).

177 178

3. Results and discussion

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3.1. Characterization of gold plated BPE

180

The morphologies of bare BPE and gold plated BPE were observed by SEM (Fig.

181

1). Bare BPE exhibited a relatively smooth surface with some imprints from rolling

182

process (Fig. 1A). After electrodeposition, AuNPs were anchored onto the cathode of

183

BPE and distributed well. As the scanning cycles of cyclic voltammetry (CV) increase

184

(Fig. 1B and 1C), large amounts of small-sized AuNPs formed, indicating that more

185

uniform gold film on the cathode of BPE (inset in Fig. 1E). Furthermore, if the

186

scanning cycles reached 30, then large amounts of irregular AuNPs formed, resulting

187

in the formation of a nonuniform gold film (Fig. 1D). Meanwhile, the influence of the

188

electrodeposition scanning cycles on the ECL intensity was also investigated (Fig. 1E).

189

The ECL intensity on gold plated BPEs (Fig. 1E, curve b to d) enhanced notably

190

compared with that on bare BPE (Fig. 1E, curve a). Meanwhile, the ECL peak voltage

191

shifted toward negative gradually (Fig. 1E, curve b to d) with the increase of

192

electrodeposition scanning cycles, confirming that external voltage for driving the

193

redox reactions on gold plated BPE was decreased. Thus, 20 scanning cycles was

194

selected for all subsequent electrodeposition experiments. Compared with bare BPE,

195

this gold plated BPE provided a 3-fold enhancement of ECL intensity, showing a

196

significant improvement of ECL sensitivity.

197 198

Fig. 2. The role of PANI in ECL performance. Experimental condition: (A) polyA59

199

is not added during the polymerization of aniline and the cathode of BPE is washed

200

with PBS after polymerization; (B) 0.5 µM polyA59 is added during the

201

polymerization of aniline and the cathode of BPE is washed with PBS after

202

polymerization; (C) polyA59 is not added during the polymerization of aniline and the

203

cathode of BPE is not washed with PBS after polymerization; (D) 0.5 µM polyA59 is

204

added during the polymerization of aniline and the cathode of BPE is not washed with

205

PBS after polymerization. Representative SEM images of washed electrode (E) and

206

unwashed electrode (F) with 0.5 µM polyA59 during the polymerization of aniline.

207 208

3.2. The role of PANI in ECL performance

209

One of the most intensively investigated enzymatic polymerizations is that of

210

aniline to yield PANI in its good conductive and electrical stability (Liu, et al., 2016).

211

The synthesis is simple, and the reaction conditions are mild. When aniline monomer

212

is added to a DNA template solution at pH 4.3, the aniline molecules become

213

protonated, and the electrostatic interaction between the protonated aniline and the

214

phosphate groups in the DNA results in close association of the protonated aniline

215

with the DNA. A water-soluble, electroactive, and electrically conductive PANI/DNA

216

complex is finally obtained (Liu et al., 1999; Ma et al., 2004). Whether the electrolyte

217

at the cathode is cleaned after the polymerization of aniline, and whether there is a

218

DNA template will have an impact on ECL performance. Fig. 2A displayed the

219

influence of whether or not the cleaning process on the decrement of ECL peak

220

voltage (∆E, ∆E = Eblank – Esample, where Eblank was the ECL peak voltage after add

221

100 ng mL-1 HRP-AFB1, Esample was the ECL peak voltage after add 100 ng mL-1

222

AFB1 sample and 100 ng mL-1 HRP-AFB1). As shown in Fig. 2A, if DNA template

223

(polyA59) is not added during the polymerization of aniline, and the cathode of BPE is

224

washed with PBS after polymerization, the ∆E is 0.03 V, and only about 100 ECL

225

peak intensity differences. It is not possible to distinguish an experimental group from

226

a blank group. As shown in Fig. 2B, If polyA59 is added during the polymerization of

227

aniline, but after the polymerization, the cathode of BPE was washed with PBS, the

228

∆E is 0.18 V, and only about 1,000 ECL peak intensity differences. It is also not

229

possible to distinguish an experimental group from a blank group. As shown in Fig.

230

2C, if polyA59 is not added during the polymerization of aniline, but after the

231

polymerization, the cathode of BPE will not be washed, the ∆E is 0.23 V, there are

232

about 3,000 ECL peak intensity differences. There is a certain difference between the

233

experimental group and the blank group. As shown in Fig. 2D, if polyA59 is added

234

during the polymerization of aniline, but after the polymerization, the cathode of BPE

235

will not be washed, the ∆E increase to 0.5 V, there are about 10,000 ECL peak

236

intensity differences. In this condition, the experimental group can be clearly

237

distinguished from the blank group. As is shown in Fig. 2E-F, the surface of the

238

washed electrode was much different from that of the unwashed electrode with 0.5

239

µM polyA59 during the polymerization of aniline. Well-distributed PANI film was

240

observed on unwashed electrode, indicating the successful polymerization of aniline.

241

Other optimized conditions for polymerization of aniline were shown in Fig. S4.

242

Therefore, 100 mM HAc-NaAc (pH 4.3, 200 mM aniline, 20 mM H2O2, 0.5 µM

243

polyA59, prepared daily) was used as PANI deposition buffer and ECL experiments

244

were performed directly without cleaning the cathode of BPE.

245 246

Fig. 3. (A) ECL-Potential response of the screen printed BPE biosensor incubated

247

with different concentrations of AFB1 (from a to i: 0 to 100 ng mL-1). (B) The

248

dependence of the concentration of AFB1 on the ECL intensity at 4.20 V. The inset

249

shows calibration curve corresponding to the value of ECL intensity as a function of

250

the logarithm concentration of AFB1. Error bars showed the standard deviation of

251

three experiments.

252 253

3.3. Determination of AFB1

254

Under optimized conditions, the BPE-ECL platform was firstly used to detect

255

AFB1 and a desirable calibration curve was achieved (Fig. 3). To show the details of

256

signal changes corresponding to a wide concentration range of AFB1, a logarithmic

257

scale was applied to concentrations of AFB1 in the graph. With increasing AFB1

258

concentration, the ECL intensity at 4.20 V gradually increased (Fig. 3A). The

259

detection limit of AFB1 (The corresponding signal change was higher than three times

260

of deviation of the signals of blank samples) was 0.033 ng mL-1 in this method, and

261

the dynamic detection range was from 0.1 to 100 ng mL-1 (Fig. 3B). Table S1

262

summarized a comparison of the analytical performances of other previous methods

263

for the detection of AFB1. This result is better or comparable to most previous reports,

264

and may satisfy the on-site analysis of AFB1.

265

3.4. Selectivity evaluation

266

The developed platform is expected to be exposed to complex samples, and so its

267

selectivity evaluation is vital prior to analysis of real samples. Here, taking the AFB1

268

assay as an example, many potential interfering species were used to investigate the

269

selectivity. AFB1 is produced by the metabolism of mycotoxins in agricultural

270

products. In these samples, however, some similar aflatoxin and other mycotoxins

271

usually exist and possibly interfere with the AFB1 determination. To evaluate this,

272

other mycotoxins (AFM1, ZEN, OTA, DON, patulin) and a mixture of mycotoxins

273

(containing AFB1, AFM1, OTA, and DON) were used as potential interfering

274

substances to further test the selectivity of the assay. As shown in Fig. 4, the presence

275

of the other kinds of mycotoxins led to negligible enhancement in the value of ECL

276

intensity compared to AFB1 and mixture at the same conditions. Therefore, the

277

proposed BPE-ECL sensor exhibited good selectivity in discriminating AFB1 and

278

other mycotoxins.

279 280

Fig. 4. Selectivity of the sensing system in the presence of different mycotoxins

281

(AFB1: 10 ng mL−1, others: 100 ng mL−1, mixture: 10 ng mL-1 AFB1 and 100 ng

282

mL−1 others). Error bars showed the standard deviation of three experiments. The

283

inset shows the ECL response curves for the corresponding targets.

284 285

3.5. Real sample measurement

286

To investigate the feasibility and applicability of the proposed method, the

287

biosensor was used to measure the levels of AFB1 in several real samples. Rice,

288

wheat, corn, sorghum, barley, and buckwheat were selected as model grains to be

289

tested. The experimental results are shown in Table 1. The detection limit of this

290

biosensor (0.033 ng mL-1) is less than the limited values of each model grains in Limit

291

of Fungal Toxins in Food, the National Food Safety Standard of China (GB

292

2761-2017) (10 ng mL-1 for rice, 5 ng mL-1 for wheat, 20 ng mL-1 for corn, 5 ng mL-1

293

for sorghum, 5 ng mL-1 for barley, and 5 ng mL-1 for buckwheat). The average

294

recovery of the biosensor and ELISA was 92.9% and 79.7% respectively. The relative

295

standard deviation (RSD, relative to an average recovery of 100%) of the biosensor

296

and ELISA was 15.24% and 24.28% respectively. Experimental results show that this

297

BPE-ECL measurement method has higher accuracy and better repetition than that of

298

ELISA, and can be practically used as a quantitative method for AFB1 detection in

299

grains samples. Table 1 Recoveries of AFB1 in grain samples for applicability of biosensor and the ELISA kit. Sample

Original

Addeda

(ng mL-1)

(ng mL-1)

ELISA

Recovery (%)

Biosensor

Recovery (%)

1

0.85

85.0

0.91

91.0

10

9.27

92.7

10.2

102

20

20.4

102

24.6

123

0.5

0.33

66.0

0.45

90.0

5

4.82

96.4

4.91

98.2

10

9.22

92.2

9.55

95.5

2

1.37

68.5

1.97

98.5

20

18.8

94.0

20.8

104

50

43.6

87.2

48.0

96.0

0.5

0.33

66.0

0.34

68.0

5

3.98

79.6

4.81

96.2

10

8.31

83.1

8.82

88.2

0.5

0.34

68.0

0.35

70.0

5

3.72

74.4

4.93

98.6

10

7.97

79.7

9.62

96.2

0.5

0.31

62.0

0.35

70.0

5

3.39

67.8

4.99

99.8

10

7.02

70.2

8.64

86.4

Rice

Wheat

Corn

Sorghum

Barley

Buckwheat a

0

0

0

0

0

0

Foundb (ng mL-1)

The bold italic data are the limited values of each grain samples in Limit of Fungal Toxins in

Food, the National Food Safety Standard of China (GB 2761-2017). b

Each data point present an average of five independent measurements.

300 301 302

4. Conclusions In summary, we developed a simple BPE-ECL assay for detection of AFB1.

303

Based on the synergistic effect of BPE and ECL, AFB1 in agricultural products could

304

be qualitatively identified. The detection interface is separated from the reporting

305

interface under the same pretreatment conditions, improve the accuracy of detection

306

and avoid false positive problems in the detection process. This strategy could also be

307

applied to fabricate other sensors for various mycotoxins detection by replacing the

308

corresponding antibody.

309

CRediT authorship contribution statement

310

Yafei Li and Xiong Xiong: Data curation, Writing - original draft. Wei Yuan and

311

Yichen Lu: Conceptualization, Methodology, Writing - review & editing. Xiaohui

312

Xiong and Yuanjian Liu: Funding acquisition, Project administration, Writing - review

313

& editing. Xiaoye Chen and Yi Li: Formal analysis, Software, Supervision.

314

Acknowledgments

315

This work was financially supported by the National Key Research and

316

Development Program of China (No. 2018YFC1602800), the National Natural

317

Science Foundation of China (No. 21804071), and Natural Science Foundation of

318

Jiangsu Province of China (No. BK20180688).

319

Notes

320 321

The authors declare no competing financial interest. Declaration of interests

322

None.

323

References

324

Chow, K.F., Mavre, F., Crooks, J.A., Chang, B.Y., Crooks, R.M., 2009. J. Am. Chem.

325

Soc. 131, 8364-8365. https://doi.org/10.1021/ja902683f

326

Feng, Q.M., Chen, H.Y., Xu, J.J., 2015. Sci. China Chem. 58, 810-818.

327

https://doi.org/10.1007/s11426-014-5295-4

328

Guerrette, J.P., Percival, S.J., Zhang, B., 2013. J. Am. Chem. Soc. 135, 855-861.

329

https://doi.org/10.1021/ja310401b

330

Guo, Z.H., Percival, S.J., Zhang, B., 2014. J. Am. Chem. Soc. 136, 8879-8882.

331

https://doi.org/10.1021/ja503656a

332

Hotta, H., Akagi, N., Sugihara, T., Ichikawa, S., Osakai, T., 2002. Electrochem.

333

Commun. 4, 472-477. https://doi.org/10.1016/S1388-2481(02)00343-0

334

Ji, J., Wang, Q.Y., Wu, H., Xia, S., Guo, H.Y., Blazenovic, I., Zhang, Y.Z., Sun, X.L.,

335

2019. Food Chem. Toxicol. 126, 106-112. https://doi.org/10.1016/j.fct.2018.12.052

336

Jiang, W.X., Wang, Z.H., Nolke, G., Zhang, J., Niu, L.L., Shen, J.Z., 2013. Food Anal.

337

Method 6, 767-774. https://doi.org/10.1007/s12161-012-9484-5

338

Khoshfetrat, S.M., Bagheri, H., Mehrgardi, M.A., 2018. Biosens. Bioelectron. 100,

339

382-388. https://doi.org/10.1016/j.bios.2017.09.035

340

Kolosova, A.Y., Shim, W.B., Yang, Z.Y., Eremin, S.A., Chung, D.H., 2006. Anal.

341

Bioanal. Chem. 384, 286-294. https://doi.org/10.1007/s00216-005-0103-9

342

Li,

343

https://doi.org/10.1016/j.talanta.2019.05.022

344

Liu, W., Cholli, A.L., Nagarajan, R., Kumar, J., Tripathy, S., Bruno, F.F., Samuelson,

345

L., 1999. J. Am. Chem. Soc. 121, 11345-11355. https://doi.org/10.1021/ja9926156

346

Liu, Y.J., Wei, M., Liu, X., Wei, W., Zhao, H., Zhang, Y.J., Liu, S.Q., 2016. Chem.

347

Commun. 52, 1796-1799. https://doi.org/ 10.1039/c5cc09800a

348

Liu, Z.Y., Qi, W.J., Xu, G.B., 2015. Chem. Soc. Rev. 44, 3117-3142. https://doi.org/

349

10.1039/c5cs00086f

350

Lv, Y.Q., Chen, S.Y., Shen, Y.F., Ji, J.J., Zhou, Q., Liu, S.Q., Zhang, Y.J., 2018. J. Am.

351

Chem. Soc. 140, 2801-2804. https://doi.org/ 10.1021/jacs.8b00515

352

Ma, Y.F., Zhang, J.M., Zhang, G.J., He, H.X., 2004. J. Am Chem. Soc. 126,

353

7097-7101. https://doi.org/ 10.1021/ja039621t

354

Munawar, H., Safaryan, A.H.M., De Girolamo, A., Garcia-Cruz, A., Marote, P., Karim,

355

K., Lippolis, V., Pascale, M., Piletsky, S.A., 2019. Food Chem. 298, UNSP 125044.

356

https://doi.org/10.1016/j.foodchem.2019.125044

357

Plana, D., Jones, F.G.E., Dryfe, R.A.W., 2010. J. Electroanal. Chem. 646, 107-113.

358

https://doi.org/10.1016/j.jelechem.2010.03.020

359

Sana, S., Anjum, A.A., Yaqub, T., Nasir, M., Ali, M.A., Abbas, M., 2019. Pak. Vet. J.

360

39, 169-174. https://doi.org/ 10.29261/pakvetj/2019.031

361

Shi, H.W., Zhao, W., Liu, Z., Liu, X.C., Xu, J.J., Chen, H.Y., 2016. Anal. Chem. 88,

362

8795-8801. https://doi.org/ 10.1021/acs.analchem.6b02204

363

Sompunga, P., Pruksametanan, N., Rangnoi, K., Choowongkomon, K., Yamabhai, M.,

364

2019. Talanta 201, 397-405. https://doi.org/ 10.1016/j.talanta.2019.04.034

365

Turner, N.W., Bramhmbhatt, H., Szabo-Vezse, M., Poma, A., Coker, R., Piletsky, S.A.,

366

2015. Anal. Chim. Acta 901, 12-33. https://doi.org/ 10.1016/j.aca.2015.10.013

367

Wang, B., Chen, Y.F., Wu, Y.Y., Weng, B., Liu, Y.S., Lu, Z.S., Li, C.M., Yu, C., 2016.

368

Biosens. Bioelectron. 78, 23-30. https://doi.org/ 10.1016/j.bios.2015.11.015

369

Wang, N.N., Feng, Y.Q., Wang, Y.W., Ju, H.X., Yan, F., 2018. Anal. Chem. 90,

370

7708-7714. https://doi.org/ 10.1021/acs.analchem.8b01610

S.,

Liu,

Y.,

Ma,

Q.,

2019.

Talanta

202,

540-545.

371

Wang, Y.Z., Ji, S.Y., Xu, H.Y., Zhao, W., Xu, J.J., Chen, H.Y., 2018. Anal. Chem. 90,

372

3570-3575. https://doi.org/10.1021/acs.analchem.8b00014

373

Wei, M., Wang, C.L., Xu, E.S., Chen, J., Xu, X.L., Wei, W., Liu, S.Q., 2019. Food

374

Chem. 282, 141-146. https://doi.org/ 10.1016/j.foodchem.2019.01.011

375

Wei, W., Li, D.F., Pan, X.H., Liu, S.Q., 2012. Analyst. 137, 2101-2106.

376

https://doi.org/ 10.1039/c2an35059a

377

Wu, M.S., Liu, Z., Shi, H.W., Chen, H.Y., Xu, J.J., 2015. Anal. Chem. 87, 530-537.

378

https://doi.org/ 10.1021/ac502989f

379

Wu, P., Hou, X., Xu, J.J., Chen, H.Y. 2014. Chem. Rev. 114, 11027-11059.

380

https://doi.org/ 10.1021/cr400710z

381

Wu, S.Z., Zhou, Z.Y., Xu, L.R., Su, B., Fang, Q., 2014. Biosens. Bioelectron. 53,

382

148-153. https://doi.org/10.1016/j.bios.2013.09.042

383

Xing, B., Zhu, W.J., Zheng, X.P., Zhu, Y.Y., Wei, Q., Wu, D., 2018. Sensor. Actuat.

384

B-Chem. 265, 403-411. https://doi.org/10.1016/j.snb.2018.03.053

385

Zhan, W., Alvarez, J., Crooks, R.M., 2002. J. Am. Chem. Soc. 124, 13265-13270.

386

https://doi.org/10.1021/ja020907s

387

Zhang, B., Fan, L.X., Zhong, H.W., Liu, Y.W., Chen, S.L., 2013. J. Am. Chem. Soc.

388

135, 10073-10080. https://doi.org/ 10.1021/ja402456b

389

Zhang, H.R., Wang, Y.Z., Zhao, W., Xu, J.J., Chen, H.Y., 2016. Anal. Chem. 88,

390

2884-2890. https://doi.org/ 10.1021/acs.analchem.5b04716

391

Zhang, X.W., Lazenby, R.A., Wu, Y., White, R.J., 2019. Anal. Chem. 91, 11467-11473.

392

https://doi.org/10.1021/acs.analchem.9b03013

393

Zhao, W.W., Wang, J., Zhu, Y.C., Xu, J.J., Chen, H.Y., 2015. Anal. Chem. 87,

394

9520-9531. https://doi.org/ 10.1021/acs.analchem.5b00497

A strategy for detection of AFB1 based on ECL-BPE assay The sensor uses cathode of BPE as functional sensing interface and anode as signal collection interface The recovery rate and accuracy of this sensor is better than that of ELISA

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.

CRediT authorship contribution statement Yafei Li and Xiong Xiong: Data curation, Writing - original draft. Wei Yuan and Yichen Lu: Conceptualization, Methodology, Writing - review & editing. Xiaohui Xiong and Yuanjian Liu: Funding acquisition, Project administration, Writing - review & editing. Xiaoye Chen and Yi Li: Formal analysis, Software, Supervision.