Nanohybrid sensor for simple, cheap, and sensitive electrochemical recognition and detection of methylglyoxal as chemical markers

Accepted Manuscript Nanohybrid sensor for simple, cheap, and sensitive electrochemical recognition and detection of methylglyoxal as chemical markers

Xiaobo Wu, Wenjuan Zhang, Cesar Morales-Verdejo, Yingying Sheng, María Belén Camarada, Li Chen, Zhong Huang, Yangping Wen PII: DOI: Reference:

S1572-6657(19)30183-3 https://doi.org/10.1016/j.jelechem.2019.03.022 JEAC 12981

To appear in:

Journal of Electroanalytical Chemistry

Received date: Revised date: Accepted date:

7 November 2018 12 February 2019 11 March 2019

Please cite this article as: X. Wu, W. Zhang, C. Morales-Verdejo, et al., Nanohybrid sensor for simple, cheap, and sensitive electrochemical recognition and detection of methylglyoxal as chemical markers, Journal of Electroanalytical Chemistry, https://doi.org/10.1016/j.jelechem.2019.03.022

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ACCEPTED MANUSCRIPT Nanohybrid sensor for simple, cheap, and sensitive Electrochemical recognition and detection of methylglyoxal as chemical markers Xiaobo Wua,†, Wenjuan Zhangb,†, Cesar Morales-Verdejoc, Yingying Shengb, María

Honeybee Research Institute, Jiangxi Agricultural University, Nanchang 330045, PR

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a

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Belén Camaradac,* Li, Chenb, Zhong Huangb, Yangping Wenb,*

b

SC

China

Institute of Functional Materials and Agricultural Applied Chemistry, Jiangxi

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

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c

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Agricultural University, Nanchang 330045, PR China

Santiago, Chile

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*Corresponding author: E-mail: [email protected] (M. Camarada),

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[email protected] (Y. Wen)

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†Xiaobo Wu and Wenjuan Zhang contributed equally to this work.

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ACCEPTED MANUSCRIPT ABSTRACT We successfully designed a nanohybrid sensor based on carboxyl-functionalized multi-walled carbon nanotubes (MWCNTs-COOH) co-functionalized with both carboxymethyl cellulose (CMC) and chitosan nanosphere (CSN) for a simple, highly-sensitive

voltammetric

recognition

and

determination

of

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low-cost,

and

structure

revealed

that

CSN-CMC

was

adhered

onto

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Morphology

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Methylglyoxal (MG) as chemical marker of New Zealand Manuka honey.

MWCNTs-COOH surface. Interactions between modifying layers and both reduction

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potential and response mechanism of MG were confirmed by theoretical calculations.

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The nanohybrid sensor displayed large electrochemical effective area, excellent electrocatalytic activity, a wide linear range of 5×10-8 – 8×10-4 mol/L, a low detection

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limit of 9.6×10-9 mol/L, and good stability as well as selectivity under the optimal

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conditions. The applicability of the fabricated sensor was assessed using three Chinese honeys and one New Zealand Manuka honey. This work will put forward a

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new strategy for electrochemical recognition and detection of MG as chemical

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markers of many agricultural production, processing, products and food and a new alternative tool for assessing economically motivated fraud and counterfeit issues in Chinese honey markets using electrochemical nanohybrid sensor. Keywords: Nanohybrid sensor, Electrochemical recognition, Methylglyoxal, Honey, Theoretical calculation, Chemical marker

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ACCEPTED MANUSCRIPT 1. Introduction Methylglyoxal (MG, Scheme S1) is derived from an important glycation intermediate that is pathologically related to macro and micro angiopathy, hyperglycemia, hypertension, insulin resistance, vascular damage and age-related

PT

complications [1]. Several studies have confirmed that higher levels of MG were

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present in diabetic patient’s plasma (MG is 7 - 8.4 μM and 2.8 - 4.2 μM in type-I and

SC

type-II diabetes, respectively) in comparison with non-diabetics (MG is around 0.1 0.7 μM in normal human plasma [2]). MG as a very useful tool for any enologist

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and/or wine analyst was evaluated malolactic fermentation and red wines [3]. MG as

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one of important chemical markers New Zealand manuka honey [4,5] was also assessed economically motivated fraud and counterfeit issues in Chinese honey

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markets. In view of the important significance of MG as chemical markers

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mentioned-above, developing a simple, fast, low-cost, and sensitive method for the MG detection in biological and food as well as honey samples is very necessary. [1],

high-performance

liquid

chromatography

[6],

gas

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Electrophoresis

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chromatography [7], and capillary electrophoresis [8] have been mainly used for detecting MG. Although these methods displayed unique advantages in accuracy and selectivity in mixtures with real samples, they are time-consuming and tedious because of their specific standard procedures, the complicated sample pretreatment, strong interference resulting from colored substances or impurities present in real samples, low sensitivity, and high-cost equipment, reagent, and operator training. Electrochemical method is a good alternative analytical method due to its merit of fast 3

ACCEPTED MANUSCRIPT analysis, low cost, simple operation, high sensitivity, good stability and selectivity. There are few reports (only Rayappan group and Chen group studied electrochemical chemo/bio-sensing of MG using chemically modified electrodes) for electrochemical measurements of MG (see Table 1 [9-15]). Earlier studies mainly

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focus on the electrochemical reduction of MG at mercury electrodes [16]. However,

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the toxicity of mercury has made this undesirable for voltammetric analysis.

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Chemically modified electrodes are probably the most suitable non-toxic alternative to mercury electrodes for electrochemically measuring MG [17]. Nanomaterials as

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excellent alternatives towards traditional materials have been employed as chemically

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modified electrodes for electrochemical chemo/bio sensors due to large surface area, excellent electrochemical properties, unique nanostructure and morphology [18-21].

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The appropriate position of Table 1

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Carbon nanotubes (CNTs) as extraordinary nanomaterials have been widely used for fabricating high-efficient electrochemical sensors owing to high conductivity,

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large specific surface area, readily modifiable surface, excellent electrocatalytic ability,

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high chemical stability, and good biocompatibility [22-27]. For instance, Chatterjee et al reported an electrochemical sensor based on single-walled CNTs [10] or platinum nanoparticles/single-walled CNTs [9]) for detecting MG in wine, beer, and human plasma samples using square wave voltammetry (SWV). My previous studies indicated that carboxyl groups functionalized CNTs (CNTs-COOH, Scheme S1) can enhance hydrogen bond interactions between functional groups and analytes (the improvement of selectivity) and improve the dispersibility of CNTs (the improvement 4

ACCEPTED MANUSCRIPT of solubility). Carboxymethyl cellulose (CMC, Scheme S1) could improve water solubility, film forming ability and adhesive property, even synergistic electrocatalytic ability, and electrochemical sensing performance [30-36]. Chitosan (CS, Scheme S1) could also improve water solubility, film forming ability, and adhesive property,

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electrocatalytic ability, and electrochemical sensing performance [33-36], CS with

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nanostructure have superior physical and chemical properties such as large surface

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area, high porosity, good tensile strength, and excellent mechanical properties in comparison whit traditional CS [33, 34].

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Inspired by these aspects mentioned-above, a facile, inexpensive, and sensitive

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SWV recognition and determination of MG as a chemical marker in different honey was performed using nanohybrid sensor based on carboxyl-functionalized

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multi-walled carbon nanotubes (MWCNTs-COOH) co-decorated with both CMC and

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CS nanosphere (CSN). Scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR) were employed for characterizing the morphology and

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structure of nanohybrid. Theoretical calculation was used for studying interactions

MG.

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between modifying layers and both reduction potential and response mechanism of

2. Experimental 2.1 Chemicals MG (40% aqueous solution) was provided from Aladdin Bio-Chem Technology Co., Ltd. Sodium salt of CMC was purchased from TCI Development Co., Ltd. CS (viscosity 50–800 mPa.s, degree of deacetylation 80–95%) was supplied by 5

ACCEPTED MANUSCRIPT Sinopharm Chemical Reagent Co., Ltd. MWCNTs-COOH (2.5% carboxy content) were obtained from Chengdu Institute of Organic Chemistry in Chinese Academy of Sciences. 0.1 M phosphate buffer solutions (PBS) with different pH were prepared from aqueous solutions of both 0.1 M Na2HPO4 and 0.1 M NaH2PO4, and pH was

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adjusted using NaOH or H3PO4. Interferents including fructose and malic acid were

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obtained from Aladdin Bio-Chem Technology Co., Ltd, they were analytical-reagent

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grade and employed without further purification. The double distilled water was used in all experiments.

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2.1 Apparatus

Cyclic voltammetry (CV) and SWV were carried out using a CHI660E

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electrochemical workstation (Chenhua Instrument Co., Shanghai, China). All

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experiments were performed with a three-electrode system including a modified GCE

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as working electrode, a KCl-saturated calomel electrode (SCE) as reference electrode and a platinum wire (Pt) as the auxiliary electrode. CT-6023 portable pH meter

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(Shanghai Lin Yu Trading Co., Ltd.) was used to measure pH values. CSN were prepared via milling using a variable frequency planet-type grinding mill (Nanjing

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Kexi Institute of Experimental Instruments, China). Comparative analysis was performed on an Agilent 1100 series HPLC system (Agilent Technologies, USA) and measured according to the national standards of China (GB/T 1109-2015. The surface morphologies were characterized by SEM with a JSM-6701F microscope (FEI Company, United States). Nicolet 5700 FT-IR Spectrometer (Thermo Nicolet Corporation, United States) was used for recording IR spectra. 2.3 Preparation of CSN-CMC-MWCNTs-COOH/GCE 6

ACCEPTED MANUSCRIPT Prior to each modification, the surface of bare GCE was cleaned mechanically by polishing carefully on a chamois using 0.05 μm Al2O3 powders until a mirror-like surface was obtained. Then sonicated by using deionized distilled water, absolute ethanol, and deionized distilled water for 5 minutes, successively, and dried at room

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temperature. The surface of GCE was also cleaned electrochemically by performing

suspension

was

prepared

by

dispersing

1mg

SC

CMC-MWCNTs-COOH

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CV for 10 cycles in the potential window -1.0 V to 1.0 V vs. SCE in PBS. 1 mg mL-1

MWCNTs-COOH in 1.0 mL CMC via the ultrasonic agitation, and CMC was

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completely dissolved in distilled water (0.3 mg mL-1). powders of CSN were obtained

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via the intermittent milling using a variable frequency planet-type grinding mill for 6 hours and were sieved through the stainless-steel screen with an ultra-fine mesh size.

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0.2% CSN solution was prepared by dispersing 0.1 g CSN fine powders in 2% acetic

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acid solution. 5 μL water-dispersible CSN-CMC-MWCNTs-COOH was drop-coated on the surface of the pretreated GCE, then dried in infrared lamp to obtain

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CSN-CMC-MWCNTs-COOH/GCE.

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2.4 Electrochemical measurements 5mL PBS containing a certain concentration of MG solution was added into a three-electrode electrochemical cell. Prior to each electrochemical test, the chemically modified electrode was placed into PBS (pH 10.0) with the stirring time of 60s. The SWV were used for the measurement of MG, all experiments were carried out at room

temperature.

The

adsorption

of

MG

on

the

surface

of

CSN-CMC-MWCNTs-COOH/GCE is removed by electrochemical cleaning in acid 7

ACCEPTED MANUSCRIPT PBS (pH 6) 2.5 Preparation of honey samples Three local honey were obtained from Honeybee Research Institute in the Jiangxi Agricultural University, New Zealand manuka honey was purchased from the

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local honey store. Four pure honey samples were diluted 20-fold using 0.1 M PBS to

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test. All sample solutions were adjusted to pH 10 with PBS. The solution was

determined using the standard addition method.

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2.6 Computational methodologies

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transferred into the electrochemical cell to be analyzed, and the content of MG were

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Geometries of MG and its derivatives were fully optimized at the density functional theory (DFT) level, as implemented in Gaussian 16 software. The Becke’s

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three parameters nonlocal hybrid exchange potential with the nonlocal correlation functional of Lee, Yang, and Parr (B3LYP) [37-39] without any symmetry restriction

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was employed, along with the Gaussian triple–ζ 6–311G(d,p) basis set. The selection of the functional and the basis set was supported by the previous work of Wang et al.

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[40], where the final optimized structural parameters indicated the reliability of this method. The neutral and cationic radical molecular structures were fully optimized without symmetry constriction and the vibrational frequencies calculations were performed at the same level of theory as the geometry optimizations to confirm that the stationary points were minima at the potential energy surface. A tight SCF convergence criterion (10−8 a.u.) was used in all calculations. The PCM solvation model [41] was implemented to optimize the neutral and cationic species in water. 8

ACCEPTED MANUSCRIPT Water was considered as implicit solvent (dielectric constant ε=78.3553) to match the experimental conditions. Redox reactions are complex electrochemical processes that involve an electron transfer from or toward an electrode surface. Computational methodologies avoid the

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explicit description of the electrode-solution interface for the estimation of

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electrochemical parameters, such as standard oxidation or reduction potentials. Based

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on a thermochemical Born-Haber cycle, depicted in Scheme 1, it is possible to predict standard oxidation potentials in solution phase. This cycle relates the free energy

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change of the redox couple in condensed phase to the gas phase, ΔGogas,I and solvation

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free energies of the reduced ΔGºS(R) and oxidized ΔGºS(R+·) species referred to the normal hydrogen electrode ΔGºNHE, which was previously established to be –4.28

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eV [42,43] in aqueous solution. All results were then referred to the SCE electrode

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(0.24 V vs NHE) [44], which was used in the experimental measurements. The appropriate position of Scheme 1

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With the aim of studying the most stable site of coordination between MG and

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the modified layer of the electrode (e.g. CSN), different coordination sites were explored by full geometry optimization of complexation reactions. Due to the size of CSN, a dimer was selected as an approximation. Optimization of complexation reactions between CSN dimer and MG were performed at the same level of theory applied to the optimization of MG and its derivatives, B3LYP/6-311g(d,p). As implemented in Gaussian 16, the charge distribution of intermolecular interactions was calculated using natural population analysis (NPA) method [45] Interaction 9

ACCEPTED MANUSCRIPT energy (Eint) was defined as the energy difference between the complex and energies of constituent monomers and was calculated using the following expression: Eint = ECSN-MG – EMG(CSN-MG) – ECSN(CSN-MG). The computation of this quantity with finite basis sets introduces error known as basis set superposition error (BSSE), because

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different numbers of basic functions were used to describe the complex and

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monomers for the same basis set. BSSE corrected interaction energies were computed

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using Boys–Bernardi counterpoise correction Scheme 1 [46]. 3. Results and discussions

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3.1 Characterization of CSN-CMC-MWCNTs-COOH

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3.1.1 SEM

CMC was generally a regular and homogeneous structure with smoothness and

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compactness [25, 28-30], while SEM images of CMC-MWCNTs-COOH indicated

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that MWCNTs-COOH were bended or entangled together tightly with each other to form an inattentive filamentous-network structure, and CMC embedded into or

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covered onto MWCNTs-COOH fibrous-network structure (image b in Fig. 1A), which

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was in accordance with our previous reports [25,26]. Surface morphology of CSN displayed a nanosphere structure (image c in Fig. 1A), while SEM images of CSN-CMC-MWCNTs-COOH indicated that both CSN and CMC was adhered onto the surface onto MWCNTs-COOH filamentous-network structure(image a in Fig. 1A), which was beneficial to form larger specific surface area and improve the solubility of MWCNTs [25,26]. The appropriate position of Fig. 1 10

ACCEPTED MANUSCRIPT 3.1.2 FT-IR spectra From FT-IR spectrum of CMC-MWCNTs-COOH (curve b in Fig. 1B), the band at 1446 and 1650 cm−1 was ascribed to C=O stretching vibrations for carbonyl groups (–COOH) of both CMC and MWCNTs-COOH. The broad peak at 3469 cm−1

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belonged to O-H stretching vibrations for hydroxy groups (–OH) of both CMC and

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MWCNTs-COOH, and the C-O stretch vibration at 1027 cm−1. CSN (curve c in Fig.

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1B) exhibits peaks of carbonyl (C=C–NHR) and amine group (–NH2) at 1437 and 1655 cm−1, respectively. The broad band due to the stretching vibration of –NH2 and

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–OH group can be observed at 3341-3526 cm−1, The broad band at 985-1133 cm−1

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were attributed to the saccharide structure of CS. For the spectrum of CSN-CMC-MWCNTs-COOH composite (curve a in Fig. 1B), the characteristic

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absorption peak of CSN disappeared, indicating that there was a strong interaction

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between CSN and CMC-MWCNTs-COOH. 3.1.3 Electrochemical effective area

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The electrochemical effective area (A) can be studied by chronocoulometry (Fig.

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S1), which was determined by plots of Q-t1/2 from the following Anson equation: Q(t) = 2nFACD1/2t1/2π-1/2 + Qdl + Qads

where n is the transferred electron number, F is the faraday constant, C is the concentration of [Fe(CN)6]3-/4-, D is the diffusion coefficient of [Fe(CN)6]3-/4(7.6×10-6 cm2/s), Qdl is the double layer charge that could be eliminated by background subtraction, Qads is the Faradaic charge. According to the linear relationships

of

Q-t1/2

as

shown

in 11

Fig.

2A,

the

A

(0.05

cm2)

of

ACCEPTED MANUSCRIPT CSN-CMC-MWCNTs-COOH/GCE is 1.29-fold increase in comparison with the bare GCE (CMC-MWCNTs-COOH/GCE with the A is 1.11-fold increase), revealing that large specific A of fabricated nanocomposite electrode. The appropriate position of Fig. 2

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3.2 Electrochemical behaviors of MG

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Fig. 2B shows electrochemical behaviors of MG at different electrodes in 0.1 M

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PBS (pH 10.0). An almost invisible reduction peak for MG at the bare GCE (curve a in Fig. 2B) indicated that the electrocatalytic ability of bare GCE toward cathodic

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reduction of MG was very poor. A wide and very weak reduction peak of MG was

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obtained when CSN was modified onto GCE surface (Fig. 2B inset), implying that the CSN/GCE displayed poor electrocatalytic ability toward cathodic reduction of MG

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due to high surface area and weak electrocatalytic ability of nanopolymer. A reduction

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peak with a remarkable enhancement of the reduction current of MG was observed at approximately -0.86 V when MWCNTs-COOH was modified on GCE surface (curve

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b in Fig. 2B), which was mainly assigned to excellent electrocatalytic ability of

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MWCNTs. More obvious reduction peaks of MG at CMC-MWCNTs-COOH/GCE were observed in comparison with that at MWCNTs-COOH/GCE(curve c in Fig. 2B), revealing that CMC interact with MWCNTs-COOH, which improved dispersibility and stability of MWCNTs-COOH in water [25, 28-30] and enhanced electrocatalytic ability

towards

the

electrochemical

reduction

of

MG.

CSN-CMC-MWCNTs-COOH/GCE displayed also a considerable enhancement of reduction

peak

current

of

MG

when 12

CSN

was

introduced

into

ACCEPTED MANUSCRIPT CMC-MWCNTs-COOH nanocomposite (curve d in Fig. 2B), which was assigned to synergistic effect such as large specific surface area, good electrocatalytic ability of CSN, and interactions between MG and functional groups of CSN. 3.3 Effect of pH effect

of

pH

on

the

electrochemical

behaviors

of

MG

at

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The

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CSN-CMC-MWCNTs-COOH in different pH values were carried out by SWV.

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Fig.3A showed the voltammetric response of 160 μM MG in the pH range from 7 to 12 (MG is very unstable in acid solutions due to its degradation or chemical reaction).

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The relationship between the anodic peak height current (Ipa) and pH values were

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presented in Fig.3C. Results showed that the peak height of MG increased with the pH increasing, then reached maximum at pH 10, finally decreased dramatically with

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the increase of pH again. Thus, pH 10 was selected as the optimized pH condition for

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further studies. In addition, the relationship between peak potentials (Epa) and pH values was showed in Fig. 3C. It is especially interesting that the Epa shifted linearly

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toward more negative potential values with the increasing the solution pH, it could be

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deduced that there were protons participated in the electrochemical process of MG. The number of protons was calculated by Nernst equation:

EP  E  ( 0

[O ] RT 2.303mRT ) ln( ox )  ( )pH  nF [R red ] nF

Where m is the proton number participating in the electrochemical process, which was obtained from slopes of Epa vs. pH as follows:

dE p / dpH   2.303mRT / nF the Ep changed linearly with the pH (Fig. 3C), the slope of dEpa/dpH plots was -0.051, 13

ACCEPTED MANUSCRIPT so the m/n ratio of MG was about 1:1. According to Bard and Faulkner, α can be given as follows:

Ep  Pp/2 

1.857 RT  44.7    mV  nF  n 

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Where Ep is the peak potential (0.88 mV), and Ep/2 is the half-wave potential (0.82 mV) that is the potential at which the current is half the peak value, αn is 0.75,

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generally, the value of α is between 0.3 and 0.7, So, from this we got the value of α to

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be 0.38. Further, the number of n in the electroreduction of MG was calculated to be 2.

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Thus, it may assume that the electrode reaction of MG was accompanied by 2 proton and 2 electrons. The aldehydic group of MG is reduced and formed hydroxyacetone

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according previous literature [47], and the electro-reduction mechanism of MG was

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given as follows:

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3.4 Theoretical calculation

The interaction between modifying layers was studied by approximating the size

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of MWCNTs by a square section of graphene (GR) 20 Å side. Considering the experimental percentage of -COOH functionalization (2.5%), the graphene layer was modified by inserting one carboxylic acid group (GR-COOH). Both structures were used to explore the binding interaction with CMC. a dimer was selected as an approximation due to the size of CMC. The interaction between GR, GR-COOH and CMC was explored by optimizing a set of 4 starting configurations. Scheme S2 depicted the most stable structures. In both cases, the interactions were mediated by 14

ACCEPTED MANUSCRIPT carboxylic and hydroxyl groups of CMC. However, the stability of GR-COOH/CMC was 43 kcal·mol-1 higher than GR/CMC, confirming the key role of the carboxylic groups on the stabilization of CMC. To explore the most stable coordination site of MG and interactions between MG

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and modified materials on the electrode surface, CSN was considered for studying

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interactions between MG and functional groups of CSN. Due to the polymeric nature

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of this material, CSN represented by a dimer to reduce the cost and time of the quantum chemical calculations. Starting geometries of CSN-MG complexes for the

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optimization were generated by placing the MG near the electron-rich sites of the

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CSN structure. The two electron rich sites of MG were considered: the terminal -CHO group and the carbonyl -C=O moiety. In the case of CSN, three sites were selected:

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the -CH2OH, the amino group -NH2 and the hydroxyl -OH moiety. Six different

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coordination pairs (a-f) were explored and summarized in Table 2. The appropriate position of Table 2

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Scheme 2 shows the optimized geometry of all complexes, in which bond

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lengths and NBO charges at selected atomic sites are depicted. All complexes exhibited an internal hydrogen bond inside the CSN structure, between the -CH2OH group and the hydroxyl belonging to the next ring. The Eint analysis indicated that the most stable CSN-MG complex corresponded to the site (b), then complexes (c) and (a). Complex (b) was the only one that presented double coordination of MG to CSN, explaining its higher stability. Complex (a) to (c), did not retain their original coordination sites, while the rest of the analyzed structures remained at the initial 15

ACCEPTED MANUSCRIPT coordination places. Structures (a) and (b), preferred coordination between the MG-C=O and the CSN-OH groups, while complex (c) resulted in higher energy interaction between MG-CHO and CSN-OH. In this case, the interaction with the CSN-NH2 group was not favored. According to these results, and considering the

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lower interaction energy of complex (d) with coordination to the CSN-NH2, MG will

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tend to coordinate to the hydroxyl groups of CSN than -NH2 or -CH2OH. On the other

SC

hand, MG will tend to interact with CSN-OH through the carbonyl group, enabling the formation of a second interaction through the MG-CHO group.

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The appropriate position of Scheme 2

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MG in water hydrates favorably to form the monohydrate (MGH) with an energetic barrier of approximately +20 kcal·mol-1 and an overall free energy change

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(DG) of -1.4 kcal·mol-1. MGH can undergo a second hydration at the ketonic group to

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form the dihydrate (MGDH). However, this second step is energetically not favored to form the MGDH with an overall DG of +2.7 kcal·mol-1, as reported by Krizner and

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coworkers [48]. The reduction mechanism of MG has been studied previously by

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Wasa [49] and Rodríguez [50]. As can be depicted in Scheme 3, two reduction paths of MG are possible. The first one associated with the reduction of MG gives the enediol structure, which rearranges to the keto form catalyzed by hydroxyl ion. The second path occurs at more cathodic potentials than the above-described reaction, thus the reduction mechanism of MG is registered as a second wave in a cyclic voltammogram that is related to the reduction of the hydrated form of MG, MGH results in the keto hydrated form, which is in equilibria with the keto structure. In 16

ACCEPTED MANUSCRIPT other words, the reduction reaction of MG needs the transference of 2 electrons and 2 protons, and electrochemical reduction path of MG and its hydrated form (MGH) is presented in Scheme 3. The appropriate position of Scheme 3

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To calculate the reduction potential of MG associated to the first reduction

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process (MG to enediol), and the reduction potential MG hydrated form (MGH), the

SC

reduced species i.e. the enediol and the hydrated keto forms were optimized at B3LYP/6–311G(d,p) level of theory. The oxidation potential of the enediol form and

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the hydrated keto, structures that experiment oxidation to give MG and MGH

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respectively, were calculated using the thermodynamic free energy Born-Haber cycle. In order to compare the electrochemical and theoretical obtained values, it is

D

necessary to transform the theoretical value adding the normal hydrogen electrode

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ΔGºNHE (–4.28 eV) in aqueous solution [42,43] and the value for SCE electrode as the reference electrode (0.24 V vs NHE) [44]. The computational oxidation potential of

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the enediol structure in water was calculated as 0.69 V, while a value of 1.81 V was

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obtained for the keto hydrated structure. Therefore, the predicted reduction potentials are -0.69 V for MG, and -1.80 V for MGH. Prior experimental results published by Wasa and Musha [49] at pH 10, reported the first and second reduction potential values of MG: -0.71 V and -1.70 V vs SCE, respectively. These values are quite close to the calculated reduction potentials by the thermodynamic cycle, confirming the proposed two-paths reduction mechanism of MG (Scheme 2). The experimental SWV study at 10 pH produced an electrochemical reduction 17

ACCEPTED MANUSCRIPT response of MG, resulting in a reduction peak value of -0.86 V. The difference between the predicted value (-0.69 V) and the experimental value could be related to the effect of the modification of the electrode surface towards overpotential of MG. The presence of new functional groups on the active surface of the electrode modified

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materials might interact with the carbonyl groups of MG, which needed the necessary

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amount of energy to produce the reduction of MG. In addition, the experimental SWV

SC

study at 10 pH indicated that the electrode reaction of MG was accompanied by 2 proton and 2 electrons, which was in accordance with the experimental value.

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3.5 Sensing performance

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3.5.1 Determination of MG

SWV signals of MG with various concentrations at nanocomposite modified

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GCE was obtained. Plots of peak currents versus MG concentrations were linear in

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the range 0.05 – 800 μM at pH 10. Fig 3B showed peak current values were increasing with the addition of the MG concentration. The good linear relationship

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between peak currents and MG concentrations was depicted in Fig 3D. Besides, the

AC

limit of detection (LOD) and limit of quantitation (LOQ) were calculated by 3 s/m and 10 s/m, respectively. Where m is the slope of the calibration curve and s is the standard deviation for the replicate measurement of the sample in the absence of analytes under the same conditions. In this work, the replication determination for 20 times was recorded in blank solution using CSN-CMC-MWCNTs-COOH/GCE, So, the calculated LOD and LOQ was 0.0096 μM and 0.032 μM, respectively. Sensitivity = m/A, so the sensitivity was 0.32 μA·μM−1·cm−2. Table 1 listed the performance 18

ACCEPTED MANUSCRIPT comparison of electrochemical chemo/bio sensors based on different chemically modified electrodes for MG measurements in previous reports. The fabricated nanocomposite sensor displayed wider linear-range and lower LOD in comparison with sensors, especially chemo-sensors, which can be used for quantitatively

PT

electrochemical determination of MG in the unknown samples.

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3.5.2 Stability of sensing electrode

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The reproducibility and repeatability of the nanocomposite voltammetric sensor based on CSN-CMC-MWCNTs-COOH/GCE were investigated. The reproducibility

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of electrode was estimated by using six different GCE modified with

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CSN-CMC-MWCNTs-COOH through the same procedure (Fig. S2A), results revealed that only slight decrease of peak current with a relative standard deviation

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(RSD) of 3.18% was observed, showing the proposed sensor has good reproducibility.

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The repeatability of CSN-CMC-MWCNTs-COOH/GCE was examined using the same procedure for 25 repetitive measurements containing 48 μM MG, and the RSD

CE

was calculated about 2.77% (Fig. S2B), indicated good repeatability of the proposed

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sensor. All those data demonstrated that CSN-CMC-MWCNTs-COOH modified electrode had a good reproducibility and repeatability for the measurement of MG. In addition, the CSN-CMC-MWCNTs-COOH/GCE was also used to assess the life-time of as-fabricated MG nanocomposite sensor. Then MG was detected again after storing them for 15 days under 4°C dry sealed condition. The peak current responses maintained 98.5% of its original current responses, implying storage stability of the as-obtained sensing electrode. 19

ACCEPTED MANUSCRIPT 3.5.3 Interferences Different substances such as amino acids (salts), organic acids (salts), and carbohydrates existed in honeys were used to evaluate the anti-interferential ability of the

as-fabricated

MG

nanocomposite

voltammetric

sensor

based

on

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CSN-CMC-MWCNTs-COOH/GCE using SWV. Most of substances did not cause any

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observable interference toward the modified electrode response due to no

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electrochemical activity or relatively far away from reduction potential of MG. As can be seen listed in Table S1, 1-fold or 10-fold of different kinds of substances were

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respectively added into MG samples under the optimal conditions, the reduction peak

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current of 48 µM MG in the presence of every interferent with two concentration gradients (48 µM, or 480 µM) was measured as follows:

where iMG and

iMG  iMixture 100% iMG

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Selectivity factor =

iMixture are response peak currents of the electrode towards MG and

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mixture (MG + interferent), The selectivity factor was used for evaluating selectivity of the fabricated sensing electrode. It could be observed clearly that there was no

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remarkable change in the peak current response (signal change below 2%) in the current response towards MG in the presence of interferents with 10-fold concentration, revealing excellent selectivity of the proposed sensor for the determination of MG. In addition, Strong alkaline conditions may also improve the sensitivity (superior electrochemical response of MG in strong alkaline solution) and interference (bad electrochemical response of most interferents in strong alkaline solution) of MG detection. 20

ACCEPTED MANUSCRIPT 3.6 Honey sample analysis Different honeys were selected as real samples for assessing the practical applicability of the as-fabricated MG nanocomposite voltammetric sensor based on CSN-CMC-MWCNTs-COOH/GCE

using

the

standard

addition

method

in

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comparison with HPLC method. We chose four different kinds of honeys for the

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quantitative analysis, all honey samples were diluted with 0.1 M PBS (pH 10.0)

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before the measurement. Table 3 listed the detection of MG content in four honey samples, the obtained results using HPLC method are in better accordance with that of

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the as-fabricated MG nanocomposite sensor, indicating that this method is feasible

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and suitable. Besides, the known amounts of MG were added into different samples, then were measured. The recovery values of all samples are in a range of 95.43% and

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104.75%, indicating that the as-fabricated MG nanocomposite sensor based on

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CSN-CMC-MWCNTs-COOH/GCE was

employed for the MG measurement in

honey samples. RSD values for the analysis of four honey samples were less than 5%

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(between 0.19% and 4.64%), revealing that the accuracy of the prepared MG

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nanocomposite sensor was acceptable in in honey and related samples. The appropriate position of Table 3

New Zealand Manuka honey presented an obvious voltammetric response in comparison with Chinese honeys (Tilia honey, Acacia honey, Manuka honey). Chinese honeys with different volume proportions were added to New Zealand Manuka honey, which resulted in different current variations (Fig. S3), indicating that the proposed method could identify the adulteration of honey, even assess 21

ACCEPTED MANUSCRIPT economically motivated fraud and counterfeit issues in Chinese honey market.

4. Conclusions A simple, low-cost, and sensitive electrochemical sensing platform based on CSN-CMC-MWCNTs-COOH was successfully constructed and employed for the determination

of

MG

in

honey

samples.

The

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voltammetric

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CMC-MWCNTs-COOH/GCE showed an irreversible electrochemical reaction toward

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MG with a well-defined electroreduction peak at approximately -0.86V and enhanced electrocatalytic activity for the reduction of MG, accompanying a two-proton and

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two-electron process, which could detect in a linear range from 5×10-8 – 8×10-4 mol/L

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with a low LOD of 9.6×10-9 mol/L under the optimal conditions. Then, the developed method was used to measure the content of MG in honey samples. Many advantages

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such as high sensitivity, wide linear range, low LOD, high repeatability and

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reproducibility, good life-time, very poor response to interferants and excellent practicability revealed that CSN-CMC-MWCNTs-COOH could be a potential

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candidate for the simple, cost-effective, sensitive and selective voltammetric

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identification of Manuka honey and assessing economically motivated fraud and counterfeit issues in Chinese honey market. ACKNOWLEDGEMENTS This study was funded by National Science Foundation of China (31660492, 31760488, 51662014), the outstanding young talent program of Jiangxi Province (20162BCB23029, 20171BCB23042), and the Scientific Research Key Project of the Jiangxi Provincial Department of Education (GJJ160351). 22

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ACCEPTED MANUSCRIPT Captions Scheme 1: Thermodynamic free energy Born-Haber cycle that was used to calculate the oxidation potential of MG. Scheme 2 The optimized structures of MG-CSN at B3LYP/6-311G level. NPA

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charges (a.u.) for selected atoms are displayed in italics and bond lengths in Å.

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Scheme 3 Electrochemical reduction path of MG and its hydrated form (MGH).

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CMC-MWCNTs-COOH (b), and CSN (c).

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Fig. 1. SEM images (A) and FTIR spectra (B) of CSN-CMC-MWCNTs-COOH (a),

Fig. 2. Q-t1/2 plots (A) of 5 mM [Fe(CN)6]3-/4- containing 0.1M KCl and SWVs (B) of

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320 uM MG in 0.1 M PBS (pH 10.0) at bare/GCE (a), MWCNTs-COOH/GCE (b), CMC-MWCNTs-COOH/GCE (c), CSN-CMC-MWCNTs-COOH/GCE (d), inset:

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SWVs (B) of 320 uM MG in 0.1 M PBS (pH 10.0) at CSN. SWV parameters:

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frequency 15 Hz, amplitude 0.025 V, quiet time 2 s, and incr E 0.004 V. Fig. 3. SWVs of 160 uM MG with different pH values (A) and different

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concentrations (B), effect of pH on anodic peak currents and anodic peak potentials

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(C), and linear relationship between peak height and MG concentrations (D) at CSN-CMC-MWCNTs-COOH/GCE.SWV parameters: frequency 15 Hz, amplitude 0.025 V, quiet time 2 s, and incr E 0.004 V. Table 1 The performance comparison of electrochemical chemo/bio sensors based on different chemically modified electrodes for sensing MG in previous reports. Table 2 Initial and final coordination sites of MG and CSN. Anchor bond distances dMG–CSN in Å. NPA derived atomic charges (a.u.) of the anchor atom qx of MG and the 30

ACCEPTED MANUSCRIPT bonded atom of CSN qx-CSN, where x is defined as O or H atom. BSSE-Corrected and -Uncorrected (in parenthesis) Interaction Energy (Eint, kcal·mol-1) for the studied complexes. Table 3. The determination of MG in different honey samples (pH 10) using the

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nanocomposite voltammetric sensor based on CSM-CMC-MWCNTs-COOH/GCE in

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comparison with HPLC method.

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ACCEPTED MANUSCRIPT Table 1 The performance comparison of electrochemical chemo/bio sensors based on different chemically modified electrodes for sensing MG in previous reports.

1 × 10-7 – 1× 10-4 1× 10-7 – 1 × 10-4 5 × 10-6 – 5 × 10-5 3 × 10-6 – 3 × 10-5 1 × 10-7 – 1 × 10-4 6 × 10-7 – 2 × 10-6 2 × 10-8 – 1 × 10-4 5 × 10-8 – 8 × 10-4

Wine and beer Human plasma Cow milk Human blood Rice Grilled chicken Grilled chicken Honeys

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Real samples

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Chemosensor Chemosensor Biosensor Chemosensor Chemosensor Biosensor Biosensor Chemosensor

Linear range (M)

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CE

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D

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Pt/SWNT/GCE SWNT/GCE Pt/CeO2/GLO1/chitosan Nanostructural V2O5 Pt/V2O5/GSH/Chitosan Pt/ZnO/GLO1 Pt/ZnO/GLO1 CSN-CMC-MWCNTs-COOH/GCE

Type

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Modified materials

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Ref. 9 10 11 12 13 14 15 This work

ACCEPTED MANUSCRIPT Table 2 Initial and final coordination sites of MG and CSN. Anchor bond distances dMG–CSN in Å. NPA derived atomic charges (a.u.) of the anchor atom qx of MG and the bonded atom of CSN qx-CSN, where x is defined as O or H atom. BSSE–Corrected and –Uncorrected (in parenthesis) Interaction Energy (Eint, kcal·mol-1) for the studied

Initial

Final

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complexes. CSN

MG

CSN

dMG-CSN

qx-MG

a b

-CHO -C=O

-CH2OH -CH2OH

c d e f

-CHO -C=O -CHO -C=O

-NH2 -NH2 -OH -OH

-C=O -C=O -CHO -CHO -C=O -CHO -C=O

-OH -OH -CH2OH -OH -NH2 -OH -OH

2.26 2.24 2.85 2.41 2.85 2.29 1.94

-0.553(O) -0.553(O) 0.128(H) -0.532(O) -0.540(O) -0.504(O) -0.546(O)

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qx-CSN

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MG

0.500(H) 0.503(H) -0.758(O) 0.499(H) 0.370(H) 0.484(H) 0.481(H)

-Eint 3.6652 (7.5192) 4.5558 (8.6822) 3.8862 (7.6887) 2.4681 (4.6824) 1.4520 (3.8982) 1.3118 (4.8107)

ACCEPTED MANUSCRIPT Table 3. The determination of MG in different honey samples (pH 10) using the nanocomposite electrocatalytic sensor based on CSM-CMC-MWCNTs-COOH/GCE in comparison with HPLC method (n = 3). Sensing method

New Zealand Manuka honey (d) Blended honey 1* (d+ a) Blended honey 2* (d + b) Blended honey 3* (d + c)

Recovery

1.05 0.77 0.2 1.64 0.56 0.26 3.55 0.71 1.47 2.88 2.69 2.85 2.70 3.16 3.77 2.74 2.71 3.33 2.38 3.13 4.37

95.25 102.94 93.33 96.79 97.13 102.66 96.84 98.10 100.42 99.06 98.27 102.91 99.16 97.65 95.43 97.94

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97.37 104.75 96.19 98.13 98.31 102.39 97.35 97.56 100.18 98.59 97.01 101.56 100.36 97.87 95.97 97.75

RSD (%)

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1.48 1.1 0.19 2.78 0.38 1.67 2.2 1.54 2.20 1.47 1.38 1.50 1.37 1.97 2.78 3.0 2.81 2.94 1.84 2.46 4.64

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Blended honey 4* (d + a + b + c)

Found (μM)

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Honey (c)

16 160 16 160 16 160 16 160 16 160 16 160 16 160 16 160

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Chinese Jujube

Recovery (%)

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honey (b)

RSD (%)

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Chinese Acacia

Found (μM)

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Chinese Tilia honey (a)

Added (μM)

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Samples

HPLC method

* New Zealand Manuka honey containing different Chinese honeys with different volume ratios.

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(%)

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Graphical abstract

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The preparation process of simple, low-cost, and sensitive nanohybrid electrochemical sensor based on CSN-CMC-MWCNTs-COOH/GCE and electrochemical recognition and determination of MG as a chemical marker of Manuka honey from Chinese honey.

35

ACCEPTED MANUSCRIPT Highlights (1) MWCNTs-COOH-CMC-CSN

nanohybrid

voltammetric

sensor

was

designed and characterized. (2) Interaction between modifying layers, reaction mechanism and reduction

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potential were verified by DFT.

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(3) This method is an alternative tool for recognition and detection of MG as

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chemical markers in honey products.

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Figure 1

Figure 2

Figure 3