Simple voltammetric analyses of ochratoxin A in food samples using highly-stable and anti-fouling black phosphorene nanosensor

Accepted Manuscript Simple voltammetric analyses of ochratoxin A in food samples using highly-stable and anti-fouling black phosphorene nanosensor Yuan Xiang, María Belén Camarada, Yangping Wen, Hao Wu, Jinyin Chen, Mingfang Li, Xiaoning Liao PII:

S0013-4686(18)31343-4

DOI:

10.1016/j.electacta.2018.06.055

Reference:

EA 32048

To appear in:

Electrochimica Acta

Received Date: 21 February 2018 Revised Date:

5 June 2018

Accepted Date: 7 June 2018

Please cite this article as: Y. Xiang, Marí.Belé. Camarada, Y. Wen, H. Wu, J. Chen, M. Li, X. Liao, Simple voltammetric analyses of ochratoxin A in food samples using highly-stable and anti-fouling black phosphorene nanosensor, Electrochimica Acta (2018), doi: 10.1016/j.electacta.2018.06.055. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Simple voltammetric analyses of ochratoxin A in food samples

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using

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nanosensor

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Yuan Xianga,c,d, María Belén Camaradab, Yangping Wen a,c,*, Hao Wua, Jinyin Chend,*,

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Mingfang Li a, Xiaoning Liaoa,c,d,*

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a

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Agricultural University, Nanchang 330045, P. R. China.

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b

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Santiago- 5750, Chile.

and

anti-fouling

black

phosphorene

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highly-stable

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Institute of Functional Materials and Agricultural Applied Chemistry, Jiangxi

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Centro de Nanotecnología Aplicada, Facultad de Ciencias, Universidad Mayor,

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c

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Education, Jiangxi Agricultural University, Nanchang 330045, P. R. China.

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d

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of Fruits and Vegetables in Jiangxi Province, Nanchang 330045, P. R. China.

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*Corresponding author: Fax: +86-791-83823320, Tel: +86-791-88537967,

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E-mail: [email protected] (Y. Wen); [email protected] (J. Chen);

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[email protected] (X. Liao)

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Collaborative Innovation Center of Postharvest Key Technology and Quality Safety

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Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of

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Abstract There are few reports on the electrochemical detection of ochratoxin A (OTA)

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using chemically modified electrode owing to the OTA poor electrochemical activity

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and strong fouling effect towards electrode surface. In this study, a novel nanosensor

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based on two-dimensional (2D) layered black phosphorene (BP) was successfully

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developed for the simple voltammetric detection of OTA in grape juice and red wine

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samples. The BP modified nanosensor showed a good linear electrochemical response

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towards OTA in a concentration range of 0.3 - 10 µg/mL with a detection limit of 0.18

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µg/mL (S/N = 3) under the optimal conditions. A mechanism for the electrocatalytic

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oxidation of OTA was proposed and verified by density functional theory calculations

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that the oxidation of OTA is an irreversible electrochemical response with an

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adsorption-controlled process, and the nitrogen atom of the amide is oxidized to N+O-

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with two protons and two electrons. The proposed electrochemical sensor

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demonstrated good electrochemical stability, superior anti-fouling property and

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excellent sensitivity towards OTA detection. An satisfactory practicability with

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recoveries between 98.8% and 103.3% was obtained in real samples determination.

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This work puts forward a new sensing platform based on the two-dimensional layered

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graphene-like nanosensor for the electrochemical determination of mycotoxins in

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edible agricultural food.

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Keywords: Ochratoxin A, Black phosphorene, Nanosensor, Oxidation mechanism,

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Theoretical calculation

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1. Introduction Ochratoxin A (OTA) is a fungal secondary metabolite mainly produced by

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Aspergillus and Penicillium species [1]. It can be found in a lager variety of

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agricultural foods such as beer, grape juice, coffee, soy milk, and wine. OTA has been

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demonstrated to be nephrotoxic, carcinogenic, teratogenic, and immunotoxic to

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human beings and was classified as the Group 2B carcinogen by the International

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Agency for Research on Cancer (IARC) [2]. Due to its high stability throughout the

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food chain and therefore hazard imposed on human beings and animals, strict

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maximum residue limits of OTA have been established by nations all over the world.

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Consequently, there is an increasing demand for developing rapid, sensitive and

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highly selective methods for the OTA determination in different food commodities.

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To date, the most frequently used methods are chromatographic ones, such as gas

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chromatography linked with mass spectroscopy, liquid chromatography coupled to

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mass spectrometry, and high-performance liquid chromatography with fluorescence

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detectors, as well as immunochemical methods including enzyme linked

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immunosorbent assays (ELISA). Chromatographic techniques offer a precise

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determination of OTA, but require time-consuming sample separation, sophisticated

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apparatus and skilled operators. ELISA related methods provide higher efficiency

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than chromatographic ones, especially in analyzing a large amount of food samples.

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For example, ELISA kits for the quantification of OTA are commercial available and

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more easy to be used, which have been employed in various of foods such as wine,

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peanuts and coffee. However, ELISA methods are susceptible to false-positive results

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due to the fact that phenolic compounds commonly found in the wine can interrupt

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antigen-antibody reaction [3, 4]. Biosensor, especially for electrochemical biosensor, seems particularly promising

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for the OTA detection in a wide range of food matrices due to its high sensitivity and

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compatibility with miniaturization [5-8]. Nevertheless, the sensitivity and lifetime of

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biosensor are seriously influenced by factors such as temperature, pH, immobilization

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support, and immunological cross-reaction. Electrochemical sensors based on

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nanomaterials can overcome the aforementioned problems due to the absence of

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biologically active species. But only a few electrochemical sensors for the

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determination of OTA have been reported using chemically modified electrode. A

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main change is that the structure of OTA is so stable that the OTA has poor

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electrochemical response towards electrode or modified electrodes. In addition, the

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application of electrochemical sensor for detecting of OTA is severly restricted by the

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strong fouling effect on electrodes driving from the oxidation products of OTA [9].

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Therefore, exploring a new nanomaterial with high electrochemical response to OTA

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and strong anti-fouling effect to its oxidation products is highly desirable.

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Two-dimensional (2D) layered nanomaterials have attracted increasing attentions

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in electrochemical sensors due to their fascinating electrochemical properties

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inherited from the ultrathin planar structures [10, 11]. Up to now, 2D layered

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nanomaterials for electrochemical sensors mainly focus on graphene because of the

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unique electronic structure, such as fast electron transfer, excellent electrocatalytic

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activity, good conductivity. However, the zero-bandgap nature hindered the wide 4

ACCEPTED MANUSCRIPT application of graphene in electronics [11-14]. Black phosphorene (BP), a new 2D

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layered semiconductor, is an excellent graphene-like electronic nanomaterial beyond

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graphene due to its lower defect density, higher carrier lifetime, faster hole mobility

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and direct bandgap. Nowadays, BP has been successfully employed to fabricate gas

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sensors [15, 16], especially to humidity sensor [17, 18]，owing to its special sensitivity

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to water. But there are few reports on BP as chemically modified material used in

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electrochemical sensors in aqueous media. A fundamental challenge is that BP

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nanosheets lose its stability under ambient conditions because it is highly reactive to

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water and oxygen, resulting in total degradation of its electrochemical properties

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eventually [19, 20]. Functionalization of BP sheets with capping layer [21], covalent

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aryl diazonium [22] and ligand surface coordination [23]can improve its stability. We

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also investigated the environmental stability of BP sheets after modified with

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PEDOT:PSS, and found that PEDOT:PSS can increase the stability of BP sheets in

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water containing oxygen in a degree [24]. More recently, Yu et al. found that

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functionalized BP with Ag+ in the presence of NMP not only improve the stability in

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aqueous conditions but also remain the original super electrochemical properties [25].

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Inspired by these work, herein, a nanosensor was fabricated using Ag+

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functionalized BP nanosheets and the sensor was employed to voltammetric analyses

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of OTA in food samples using differential pulse voltammetric (DPV) (Scheme 1).

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The BP modified sensor shows good response and stronge antifouling towards OTA

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oxidation. In addition, a possible mechanism for the electrocatalytic oxidation of

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OTA was proposed (Scheme 1), and confirmed for the first time by the density 5

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functional theory calculations imitating the aqueous solution conditions.

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2. Experimental

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2.1.Chemicals and reagents OTA (purity, 98%) was purchased from Shanghai Aladdin Bio-Chem Technology

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Co. Ltd (Shanghai, China). BP nanosheets, prepared by mechanical exfoliation and

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functionalized in NMP solution containing Ag+, was presented by Nanjing XFNANO

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Materials Tech Co. Ltd (Nanjing, China) and used as received. 0.1 mol/L phosphate

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buffer saline (PBS) of various pH values were prepared by mixing of stock solution of

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0.1 mol/L NaH2PO4, Na2HPO4, NaCl. Standard stock solution (10-3 µg/mL) of OTA

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was prepared by dissolving OTA in absolute ethanol. All other chemicals were

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analytical grade and obtained from Shanghai Vita Chemical Reagent Co. Ltd. Milli-Q

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water (18.2 MΩ/cm) was used in all experiments.

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2.2. Apparatus

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All electrochemical measurements were performed using a CHI660E

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electrochemical workstation (Chenhua Instrument Company, Shanghai, China) in a

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three-electrode system, consisting of GCE or modified GCE as the working electrode,

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a platinum wire as the counter electrode, and a saturated calomel electrode (SCE) as

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the reference electrode. The pH values of buffer solutions were determined with a

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CT-6023 portable pH meter (Shanghai Jingmi Instrument, China). The surface

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morphologies were characterized by Scanning Electron Microscopy (SEM) with a

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JSM-6701F microscope (JEOL Ltd, Japan) and Transmission Electron Microscope

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(TEM) with a TECNAI G2 F20 S-TWIN microscope (FEI, America). X-ray

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ACCEPTED MANUSCRIPT photoelectron spectroscopy (XPS) was performed on the Thermo Fisher ESCALAB

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250Xi XPS with an Al Kα X-ray source. Raman scattering was conducted on the

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Horiba Jobin–Yvon LabRam HR-VIS high-resolution confocal Raman microscope

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equipped with a 532 nm laser as the excitation source.

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2.3. Preparation of modified electrode

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The bare GCE (3 mm in diameter) was firstly polished on a chamois leather with

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0.05 µm alumina slurry, then cleaned successively with deionized water, absolute

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ethanol, and deionized water by sonication for 5 min, respectively, finally dried in a

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high purity N2 stream at room temperature. The BP modified GCE was obtained by

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drop coating of 10 µL BP suspension on the bare GCE surface and dried under an

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infrared lamp. To get well dispersed suspension, the BP nanosheets were ultrasonic

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for 30 min before modification.

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2.4. Electrochemical measurements

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The electrochemical behavior was characterized by Cyclic voltammetry (CV) and

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differential pulse voltammetry (DPV) in the potential range from 0.5 V to 1.2 V. The

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electrochemical impedance spectroscopy (EIS) were carried out in 5 mmol/L

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[Fe(CN)6]3-/4- containing 0.1 mol/L KCl at the open circuit potential with a frequency

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range from 1 Hz to 105 Hz and an amplitude of 5 mV. The quantitative determination

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of OTA was performed by DPV with the potential range 0.5 – 1.2 V with the pulse

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amplitude of 0.05 V and the pulse width of 0.05 s, the desired concentrations of OTA

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were added to 5.0 mL PBS solution. All experiments were performed at room

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temperature.

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2.5. Computational details Geometries were fully optimized at the density functional theory level, as

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implemented in Gaussian16 software. The Becke’s three parameter nonlocal hybrid

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exchange potential with the nonlocal correlation functional of Lee, Yang, and Parr

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(B3LYP) [26-28] without any symmetry restriction was employed, along with the

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Gaussian triple–ζ 6–311G (d, p) basis set. The molecular structures were fully

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optimized without symmetry constriction and the vibrational frequencies calculations

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were performed at the same level of theory as geometry optimizations to confirm that

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stationary points were minima at the potential energy surface (PES). A tight SCF

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convergence criteria (10−8 a.u.) was used in all calculations. Charge distribution of

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intermolecular interactions was calculated using the natural population analysis (NPA)

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method [29], as implemented in Gaussian16. Fukui (f) functions are important local

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descriptor of site selectivity [30]. The fukui function for electrophilic attacks (f –) is

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related to the frontier molecular orbitals through their respective electronic densities

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[31]. Plots of f – describe sites with higher electron density which are more susceptible

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of oxidation.

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2.6. Preparation of real samples

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The grapes and the beer were purchased from a local market. The grape juice

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was obtained by smashing suitable amounts of grapes into pulp by homogenizer and

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then filtrated with a 0.45 µm nylon filter membrane. Both grape juice and beer

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samples were adjusted to pH 6.0 with 0.1 mol/L NaH2PO4 and Na2HPO4 before

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detection. The contents of OTA in each sample were determined by DPV under the 8

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3. Results and discussion

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3.1.Characterizations of BP modified electrode

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3.1.1. Surface Characterizations

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Surface morphologies and microstructures of BP nanosheets were characterized

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by SEM and TEM. As shown in Fig. 1A, BP sheets have the typical lamellar structure

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with average width of about 1 µm and average length more than 4 µm. The TEM

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image of BP in Fig. 1B further reveals that the BP nanosheets are few-layer and

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lamellar structures. Fig. 1C shows the obtained Raman spectra for thin BP nanosheets.

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The peak observed at 364 cm−1 corresponds to the A1g , while peaks at 440 and 468

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cm−1 can be ascribed to in-plane modes B2g and A 2g modes of few-layer BP,

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respectively [32].Compare to the BP bulk reported previously, the A1g , A2g and B2g

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modes of the BP show few blue-shift [33].This phenomenon is quite similar to the

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mode shift of 2D layered nanomaterials like MoS2 [34], graphene [35] due to thin

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thickness or small lateral dimensions. These results indicate that the crystalline

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structure of the BP nanosheets remains preserved after exfoliation and sample

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preparation process, suggesting BP nanosheets keep the inherent merits form bulk

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samples. The chemical and electronic states of the element for the BP are

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characterized by X-ray photoelectron spectroscopy (XPS). As presented in Fig. 1D,

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the BP nanosheets have the high intensity peaks of P 2P3/2 and P 2P1/2 at 129.7 eV and

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130.5 eV, respectively, corresponding to the phosphorus atomic states [36], which are

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in well agreement with previous work [21]. The peak at 133.0 eV, which is much less

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ACCEPTED MANUSCRIPT intensity, is attributed to the cation-π interaction between Ag+ and BP [25]. However,

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almost no peak at 134 eV characteristic of oxidized phosphorus i.e. PxOy is observed,

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indicating the stability of BP nanosheets in ambient environment has been greatly

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improved via functionalizing with NMP solution containing Ag+ [25].

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3.1.2. EIS and CVs of BP modified electrode

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EIS and CVs were used to reveal electrochemical properties of BP modified

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GCE, performed in 5 mmol/L [Fe(CN)6]3-/4- probe solution containing 0.1 mol/L KCl.

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(Fig. S1). The Nyquist plots were fitted using Randle equivalent circuits (insert in Fig.

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S1A). The charge transfer (Rct) of BP modified electrode is calculated to be 109.9 Ω

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which is lower than 198.9 Ω that of bare electrode, showing that BP modification

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increases the rate of electron transfer. In addition, the peak-to-peak separation

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between cathodic peak and anodic peak potential of BP modified electrode is 82 mV

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which is smaller than 146 mV that of the bare GCE (Fig. S1B), further confirming

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good conductivity of BP (Fig. S1B). In addition, the peak current (Ip, a) of BP

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modified electrode is 107 µA larger than 95 µA that of the bare GCE, revealing that

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BP improved the catalytic activity.

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3.1.3. Electrochemical stability of BP modified GCE

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The electrochemical stability of BP/GCE in aquoues media containing oxygen

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was investigated by continuous CVs (in Fig. S2). The RSD of 40 successive

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measurements for Ip, a and Ip, c of BP/GCE (Fig. S2A) in 5 mmol/L [Fe(CN)6]3-/4-

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containing 0.1 mol/L KCl are 2.81% and 1.95%, respectively, indicating no

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significant loss of electrochemical activity for BP modified GCE. The successive CVs 10

ACCEPTED MANUSCRIPT of BP/GCE for 40 cycles in 0.1 mol/L PBS (pH 6.0) were also recorded. (in Fig. S2B),

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showing slight differences between the first cycle and those following. Moreover, the

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integral area of the CV decreases only 1.38% after 40 cycles, demonstrating excellent

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electrochemical stability of BP modified electrode in aqueous solution containing

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oxygen. These results imply that the BP/GCE in aqueous solution containing oxygen

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had good cycle stability and high degree of the reversibility in the repetitive

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charge/discharge cycling.

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3.1.4. Electrochemical effective surface area of BP modified GCE

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The electrochemical effective surface area of electrode was studied by

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chronocoulometry in 5 mmol/L [Fe(CN)6]3-/4- containing 0.1 mol/L KCl (Fig. S3A),

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which was determined by the plot of Q-t1/2 from the following Anson equation [37,

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38]:

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Q(t) = 2nFACD1/2t1/2π-1/2 + Qdl + Qads

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where A is the electrode effective area, n is the transferred electron number, F is the

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faraday constant, C is the concentration of the [Fe(CN)6]3-/4-, D is the diffusion

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coefficient of the [Fe(CN)6]3-/4- solution (7.6×10-6 cm2/s) [39], Qdl is the double layer

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charge which could be eliminated by background subtraction, Qads is the Faradaic

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charge. According to the linear relationships of Q-t1/2 as shown in Fig. S3B, the

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effective surface area is calculated to be 0.062 cm2 and 0.047 cm2 for the BP/GCE and

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the bare GCE, respectively. The results reveal that the BP can increase the effective

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surface area of the electrode.

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3.1.5. Adsorption capacity of BP modified electrode

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The adsorption capacity (Ts) of the working electrode was obtained by

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chronocoulometry (Fig. S3C and D) based on the equation as follows:

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Qads =nFATs

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the Ts of the BP/GCE and the bare GCE was calculated to be 8.94×10-10 mol/cm2 and

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1.90×10-10 mol/cm2, respectively. Obviously, the Ts value of the BP/GCE is much

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larger than that of the bare GCE, indicating that BP/GCE has better adsorption

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capacity towards target molecules.

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3.2. Electrochemical behaviors of OTA

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Fig. 2 shows the CV and DPV responses of 50 µg/mL OTA at different electrodes

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in 0.1 mol/L PBS (pH 6.0). An oxidation peak is presented in 0.93 V, but no reduction

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peak is observed for both CV curves in the potential range from 0.5 V to 1.2 V,

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suggesting an irreversible electrochemical oxidation of OTA. The peak current (Ip,a) of

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the BP modified GCE increase obviously compared to that of the bare electrode,

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indicating that BP/GCE has better electrocatalytic activity towards the electroxidation

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of OTA. In addition, the Ip, a of DPV remains stable after undergoing several scanning

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at BP/GCE as can be seen from the Fig. S4A, indicating good anti-fouling properties

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towards OTA reaction. However, the golden nanopaticle modified GCE shows rapid

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response degradation towards the OTA oxidation, showing the electrochemical

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reaction has high poison effect on golden particle modified GCE. According to

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Oliveira [9], the fouling effect or poison effect is stemmed from the strong adsorption

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ability by the OTA oxidation product which results in blocking of the electrode

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surface. Meanwhile, the result indicates that the BP modified electrode can avoid the

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ACCEPTED MANUSCRIPT contamination effectively from electrooxidation products of OTA. The anti-fouling

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effect for BP/GCE may be attributed to the high surface area or the strong cation-π

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interaction between Ag+ and BP reducing the adsorption opportunity of BP to OTA

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oxidation product. The actual anti-fouling mechanism for BP nanosheets towards

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OTA needs to be studied further.

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3.3. Parametric optimization

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3.3.1. Effect of pH

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The pH influence on the electrocatalytic oxidation towards OTA at BP/GCE in

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0.1 mol/L PBS was studied by DPV in the range of pH 2.0 - 7.0 (Fig. 3). The peak

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currents increase gradually with the pH value increasing from 2.0 to 6.0, and the peak

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currents decrease when the pH value exceeded pH 6.0 (Fig. 3 inset). The maximum

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value of peak current is found at pH 6.0, therefore, pH 6.0 was selected for

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subsequent experiments. In addition, peak potentials shift negatively with the increase

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of pH, revealing that the protons are involved in the electrocatalytic process in acid

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electrolytes, in agreement with the previous results [9].

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3.3.2. Effect of scan rates

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To acquire kinetic parameters of the electrocatalytic oxidation of OTA at

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BP/GCE, the cyclic voltammograms corresponding to various scan rates for the

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BP/GCE were investigated (in Fig. 4). The peak currents increase linearly with the

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increasing of scan rates, (Fig. 4 inset), and the peak potentials shifte positively,

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indicating that the reaction of OTA is an typical adsorption-controlled process.

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3.4. Mechanism for electrocatalytic oxidation of OTA 13

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3.4.1. Number of electrons and protons α is the transfer coefficient, which can be calculated by Butler and Volmer equation, the equation is simplified as follows:

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where i is the current density, i0 is the exchange current density, η is the overpotential,

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and F, R, T have their traditional significance. The plot of log i versus η showed in Fig.

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S5 inset is Tafel plot, which is essential for evaluating kinetic parameters. Two

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branches, one is an anodic branch with slope of (1-α)F/2.3RT, and the other one is a

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cathodic branch with slope of -αF/2.3RT is observed. Fig. S5 shows the linear

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equation between log i and η from the cathothic branch. The value of the slope is

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-7.06, so α is calculated to be 0.42.

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RT RTk 0 RT ) ln( )+( ) ln v αnF αnF αnF

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where n is the number of electrons in electrode reaction, Fig. 5A demonstrates the

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relationship between the peak potential and lnv, the number of electrons is calculated

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from the slope of the linear regression equation in Fig. 5A, and the value of αn is 0.78,

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therefore the proton number is determined to be 2.

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In addition, the number of protons is calculated by Nernst equation:

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RT [Ox ] 2.303mRT ) ln( )+( ) pH αnF [Re d ] αnF

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which can be determined from slopes of Ep vs. pH, the Ep changes linearly with the

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pH (Fig. 5B), and the linear relationship can be expressed as: EOTA(V) = -0.06037pH 14

ACCEPTED MANUSCRIPT + 1.235 (R2 = 0.9911), the slope of Ep vs. pH plots are 60 mV per pH. Thus the m/n

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ratio is approximately 1, indicating that equal number of electrons and protons

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involved in the electrochemical reaction, therefore the number of the proton is

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calculated to be 2.

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3.4.2. Theoretical calculation for electrooxidation site of OTA

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The ochratoxin structure was optimized in base of a crystallographic structure

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reported by Bredenkamp and coworkers [41]. At pH 6.0 ochratoxin is ionized,

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presenting a depronated carboxylic group and a protonated phenol (pKa1 = 4.4, pKa2 =

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7.3, respectively [42]). Fig. 6A shows frontier orbital plots, HOMO and LUMO of the

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optimized structure at B3LYP/6-311g (d, p) level. Because of the equilibrium between

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the protonation and deprotonation of the carboxylic acid, two states were considered.

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In the case of the neutral molecule, the most stable structure presented two hydrogen

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bonds. The first one, related to the phenolic proton H-bonded with the lactone

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carbonyl and the second related to the amide proton bonded to the phenolic oxygen.

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This structure is in agreement with previous reports that analyzed the crystal structure

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of ochratoxin by IR spectra [43]. The HOMO frontier orbital localizes high electron

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density on the aromatic phenyl ring, carboxylic group and on the amide group. In the

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case of the anionic structure, the most stable geometry involved a rotation of the

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carboxylic acid group to avoid electronic repulsion with the carbonyl group of the

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amide. The HOMO was mainly located on the –COO- group due to its higher

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electronic density, and also on the amide. LUMO, in neutral and anionic state, was

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delocalized around the phenol. Taking into account both structures and the fact that

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carboxylic acids can only be oxidized to CO2, the most probable site for the oxidation

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of OTA will be the nitrogen atom of the amide, which can be oxidized to N+O-. Besides, the natural atomic charges (NC) were also calculated and are shown in

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Fig. 6B It can be observed from this analysis, that the sites with greater electronic

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population in the neutral state are the nitrogen atom (-0.668) of the amide group, the

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oxygen atom (-0.698) of the phenol and the oxygen atoms of the carboxylic moiety. In

7

the deprotonated ochratoxin structure, again most of the charge was concentrated on

8

the carboxylic group and the amide. This evidence suggests that the most susceptible

9

regions to oxidation, i.e. the most reactive sites for an oxidation, are the nitrogen atom

11

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of the amide and the –OH group of the phenolic ring.

To get more insight about the oxidation process, the fukui (f –) function was also –

plotted (Fig. S6). f

13

The site with the highest f – value denotes higher electron density and therefore, is the

14

region of the molecule that will suffer oxidation with upper probability. In the case of

15

the protonated ochratoxin, the f

16

carboxylic acid, and the amide group.

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function is delocalized around the phenol, the

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17

is related to the region that might suffer an electrophilic attack.

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All these evidences demonstrate that the most probable site for the oxidation of

18

OTA is the nitrogen atom of the amide that can be oxidized to N+O-, and the hydroxyl

19

group of the phenol, which can be oxidized to a carbonyl group.

20

3.4.3. Electrooxidation mechanism of OTA

21

Since the electrochemical behaviors of OTA reveal that the oxidation of OTA is an

22

irreversible electrochemical process with two electrons and two protons. Furthermore, 16

ACCEPTED MANUSCRIPT the most probable site for the oxidation of OTA based on the density functional theory

2

calculations is the nitrogen atom of the amide oxidized to N+O-, or the hydroxyl group

3

of the phenol oxidized to a carbonyl group. According to previous work, the single

4

hydroxyl group in the phenol electrooxidized to a carbonyl group proceeds with only

5

one proton and one electron process [44, 45], while the amino group in carbendazim

6

electrooxidized to N+O- undergoes with two protons and two electrons [46, 47].

7

Therefore, it can be concluded that the electrooxidation site of OTA is most likely to

8

be the nitrogen atom of the amide in OTA, which is depicted in Scheme 2.

9

3.5. Determination of OTA

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Fig. 7 displays DPV responses of different OTA concentrations at the BP

11

modified electrode. As shown in Fig. 7A, the peak currents of OTA increase with

12

increasing OTA concentration and display a good linearity in the concentration range

13

of 0.3 - 10 µg/mL (Fig. 7B), following the linear regression equation IOTA (µA) =

14

0.2345C + 0.095 (R2 = 0.999). The LOD for OTA is calculated to be 0.18 µg/mL,

15

using the equation LOD = 3Sd/b, where Sd is the standard deviation of 20 times

16

electrochemical responses for blank solution and b is the slope of the calibration curve.

17

This result indicates that graphene-like two-dimensional layered BP is an excellent

18

sensing material for the development of electrochemical sensing platform.

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The determination of OTA at proposed sensor was compared with other sensors

20

reported previously, and the results are listed in Table 1. Note that only a few

21

electrochemical sensors based on chemically modified electrodes have been reported,

22

biosensors were thus included for diversity consideration. The BP/GCE displays a 17

ACCEPTED MANUSCRIPT higher sensitivity than that of Edge-plane pyrolytic graphite electrode [48].

2

Nevertheless, the proposed sensor shows lower sensitivity than that of biosensors [49,

3

50], which can be explained by the high specificity between antigen and antibody

4

reaction in biosensors. But false-positive result is an another question deserve to be

5

considered in practical application. In addition, the golden nanoparticle based sensor

6

seems have high sensitivity towards OTA detection [51]. But our experiment result

7

shows that golden nanoparticles have no antifouling effect towards OTA oxidation.

8

The combination of BP and gold nanoparticle may present a promising sensor

9

characterized with ultrahigh sensitivity and high anti-fouling effect in the

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10

determination of OTA.

11

3.6. Repeatability, reproducibility, selectivity

The repeatability of the BP modified electrode for the voltammetric response of

13

0.5 µg/mL OTA were estimated by 20th successive measurements. The RSD of the

14

repeatable peak current is 1.62% (Fig. S7A), showing an excellent repeatability of

15

BP/GCE.

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The reproducibility of the BP nanosensor for the determination of 0.5 µg/mL

17

OTA was measured at six different electrodes under the same conditions, respectively.

18

The voltammetric peak height of all electrodes were almost the same, the relative

19

standard deviation (RSD) is 3.37% (Fig. S7B), indicating the proposed method has

20

good reproducibility.

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Possible interferences for the voltammetric detection of OTA at BP/GCE were

22

investigated by adding various foreign species into a fixed amount of OTA (1 µg/mL) 18

ACCEPTED MANUSCRIPT containing 0.1 mol/L PBS (pH 6.0) (Table S1). The results indicates that the

2

concentrations of 50 times of alcohol, D-Fructose, tartaric acid, L-ascorbic acid, citric

3

acid, DL-malic acid, sucrose and L-lysine can not affect the determination of OTA

4

(RSD ˂ 5%), indicating that the obtained sensor has good anti-interference ability.

5

3.7. Practical Application

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To evaluate the feasibility and validity, the proposed BP nanosensor was used to

7

detect the OTA in beer and grape juice samples by using the standard addition method.

8

All results are presented in Table 2, it can be found that the recoveries are in the range

9

of 98.8% - 102.7% in beer samples and 101.3% - 103.3% in grape juice samples,

10

respectively. In addition, the samples were also analyzed by HPLC method and the

11

results were compared with the those achieved by DPV using the proposed sensor. It

12

is observed that the concentrations of OTA in real samples determined by the

13

proposed sensor are basically equal to those of the conventional HPLC method. The

14

RSD values for both methods are less than 5%, indicating that BP nanosensor has

15

potential practical application in real sample analyses.

16

4. Conclusions

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A novel BP electrochemical nanosensor was fabricated and employed for the

18

voltammetric analyses of OTA in beer and grape juice samples using differential

19

pulse voltammetric method. BP modified electrode displays good electrochemical

20

stability, good electrocatalytic activity, and superior anti-fouling property. The BP

21

nanosensor can sensitively detect OTA in a linear concentration range of 0.3 – 10

22

µg/mL with a LOD of 0.18 µg/mL. The proposed method also had good repeatability, 19

ACCEPTED MANUSCRIPT reproducibility and anti-interference, which was used to determine the beer and grape

2

juice samples with satisfactory recoveries of 98.8% - 102.7% and 101.3% - 103.3%,

3

respectively. The mechanism for the electrocatalytic oxidation of OTA is an

4

irreversible electrochemical response with an adsorption-controlled process, and the

5

nitrogen atom of the amide in OTA is oxidized to N+O- with the number of both two

6

protons and two electrons. Further improvement of the highly-stable BP modified

7

electrode and electrochemical determination with ultrahigh sensitivity and selectivity

8

of OTA is also in progress.

9

Acknowledgments

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The work was supported by the National Science Foundation of China

11

(31660492, 51662014), The Outstanding Young Talent Program of Jiangxi Province

12

(20171BCB23042), Postdoctoral Science Foundation in China (2015M571987),

13

Special Funds for Postdoctoral Research Funds in Jiangxi Province (2015KY44),

14

Jiangxi Provincial Department of Education (GJJ160351，GJJ170274), and Fondecyt

15

Chile Project Regular (1180023).

16

References

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Detection of Patulin and Ochratoxin A on Well‐Defined Carbon Electrodes,

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simplified

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carboxymethylated dextran modified gold surface for ochratoxin A analysis,

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[51] P. Norouzi, B. Larijani, M. Ganjali, Ochratoxin A sensor based on

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coulometric FFT cyclic voltammetry, Int. J. Electrochem. Sci. 7 (2012) 24.

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ACCEPTED MANUSCRIPT Figure captions

2

Scheme 1 The preparation process of BP/GCE nanosensor and its voltammetric

3

determination of OTA in grape juice and red wine samples.

4

Scheme 2 The electrooxidation mechanism of OTA.

5

Fig.1 SEM (A) and TEM (B) images of BP film; (C) Raman spectra, the A1g , A2g and

6

B2g modes observed in BP; (D) XPS spectra, the peak P 2P3/2 and P 2P1/2 correspond

7

to BP.

8

Fig. 2 CVs of 50 µg/mL OTA in 0.1 mol/L PBS (pH 6.0) at bare GCE, BP/GCE, and

9

the inset of corresponding DPVs.

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Fig. 3 DPVs of 50 µg/mL OTA at BP/GCE with different pH values; Inset: The

11

influence of pH on the peak current of 50 µg/mL OTA.

12

Fig. 4 CVs of BP/GCE in 0.1 mol/L PBS (pH 6.0) containing 50 µg/mL OTA at scan

13

rates from a to g with 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3 V s-1 respectively.

14

Inset: The linear relationship of OTA between Ipa and v.

15

Fig. 5 (A) The linear relationship of OTA between Epa and lnv; (B) The linear

16

relationship of OTA between Epa and pH.

17

Fig. 6 (A) The graphical representation of HOMO and LUMO of OTA in protonated

18

and deprotonated state; (B) Charges at selected atoms calculated for the protonated

19

and deprotonated state of OTA.

20

Fig. 7 (A) DPVs of OTA with different concentrations at BP/GCE. OTA

21

concentrations from a to h with 0.3, 0.5, 0.7, 1, 3, 5, 7 and 10 µg/mL, respectively. (B)

22

The linear relationship between peak currents and OTA concentrations.

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ACCEPTED MANUSCRIPT Table. 1 Comparison of the proposed sensor with other methods.for the determination

2

of OTA

3

Table. 2 Determination of OTA in real samples and its spiked recovery by the

4

proposed BP nanosensor and by the coventional HPLC (n = 3).

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ACCEPTED MANUSCRIPT Table.1 Comparison of the proposed sensor with other methods.for the determination of OTA Linear range (µg/mL)

LOD (µg/mL)

Recovery(%)

EPPG electrode MBs/CSPE immunosensor CMD/SPGE immunosensor AuNPs/RGNS-IL/GCE BP/GCE

0.8 – 2.0 1.3×10-3–1.5×10-1 1.0×10-4–1.0×10-2 4.0×10-4–8.0×10-2 0.3–10.0

0.8 3.2×10-4 5×10-4 8.8×10-5 0.18

– 80.0%–100.0 % – – 98.8%–103.3%

Magnetic

beads,

CSPEs:

Carbon

screen-printed

[48] [49] [50] [51] This work

electrode,

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MBs:

Ref.

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CMD:

carboxymethylated dextran, SPGE: Screen-printed gold electrode, AuNPs: Gold

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Edge-plane pyrolytic graphite, GCE: Glass carbon electrodes.

ACCEPTED MANUSCRIPT Table. 2 Determination of OTA in real samples and its spiked recoveries by the proposed BP nanosensor and by the coventional HPLC (n = 3).

Beer

1.00 10.00 1.00 10.00

DPV at BP/GCE

HPLC

Found µg/mL

Recovery %

RSD (n=3) %

Found µg/mL

0.99 ± 0.04 10.27 ± 0.28 1.03 ± 0.04 10.13 ± 0.36

98.8 102.7 103.3 101.3

4.01 2.71 3.73 3.57

1.00 ± 0.02 10.19 ± 0.01 1.070 ± 0.003 10.08 ± 0.009

Recovery %

RSD (n=3) %

100.1 101.9 106.6 100.8

1.65 0.12 0.33 0.10

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Grape juice

Spiked µg/mL

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Scheme 1

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Scheme 2

A1g

400 450 500 Raman Shift (cm-1)

550

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B2g

C Intensity (a.u.)

A2g

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136

P 2P3/2

P 2P1/2

+

P-Ag

134 132 130 Binding Energy (eV)

128

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BP / GCE Bare GCE

12

I / µA

9

I / µA

30

6 3 0 0.4

20

0.6 0.8 1.0 E / V vs. SCE

1.2

10 0 0.6

0.8

1.0

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20 I / µA

6 5 4

12

pH 7

3 2

8

3

4

5

6

7

pH

0.6

0.8

SC

4 0 0.4

pH 2

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I / µA

16

7

1.0

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30

14.0

y = 10.34 x + 8.731 2 R = 0.9941

12.6 11.9

18

g

11.2

12

0.21 0.28 0.35 0.42 0.49 -1 v/Vs

a

6 0 0.6

0.8

1.0

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E / V vs.SCE

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Fig. 4

1.2

SC

0.4

RI PT

I / µA

24

I / µA

13.3

ACCEPTED MANUSCRIPT 1.12

A

0.94 0.92

y = -0.06037 x + 1.235 2 R = 0.9911

1.05 0.98 0.91 0.84

0.90 -3.5

-3.0

-2.5 -2.0 -1.5 ln(v / V s-1)

-1.0

2

3

4

5

pH

EP

TE D

M AN U

SC

Fig. 5

AC C

B

RI PT

0.96

y = 0.0328 x + 1.031 R2 = 0.9944

E / V vs. SCE

E / V vs. SCE

0.98

6

7

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

Fig. 6

ACCEPTED MANUSCRIPT 2.5

A h

2.0

2.4

I / µA

I / µA

3.2 a

1.6

1.5

B y = 0.2345 x + 0.09497 R2=0.9990

1.0 0.5

0.8

0.0

0.6

0.8 1.0 E / V vs. SCE

1.2

0

4 6 COTA / µg mL-1

AC C

EP

TE D

M AN U

SC

Fig. 7

2

RI PT

4.0

8

10