copper oxide nano-flowers based acetylcholinesterase biosensor for sensitive detection of organophosphate pesticides

copper oxide nano-flowers based acetylcholinesterase biosensor for sensitive detection of organophosphate pesticides

Accepted Manuscript Title: 3D Graphene/Copper Oxide Nano-flowers Based Acetylcholinesterase Biosensor for Sensitive Detection of Organophosphate Pesti...

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Accepted Manuscript Title: 3D Graphene/Copper Oxide Nano-flowers Based Acetylcholinesterase Biosensor for Sensitive Detection of Organophosphate Pesticides Authors: Jing Bao, Ting Huang, Zhaonan Wang, Han Yang, Xintong Geng, Guoli Xu, Mickey Samalo, Mina Sakinati, Danqun Huo, Changjun Hou PII: DOI: Reference:

S0925-4005(18)31753-2 https://doi.org/10.1016/j.snb.2018.09.118 SNB 25424

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

10-5-2018 13-9-2018 27-9-2018

Please cite this article as: Bao J, Huang T, Wang Z, Yang H, Geng X, Xu G, Samalo M, Sakinati M, Huo D, Hou C, 3D Graphene/Copper Oxide Nano-flowers Based Acetylcholinesterase Biosensor for Sensitive Detection of Organophosphate Pesticides, Sensors and amp; Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.09.118 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.

3D Graphene/Copper Oxide Nano-flowers Based Acetylcholinesterase Biosensor for Sensitive Detection of Organophosphate Pesticides Jing Bao a,1, Ting Huang a,1, Zhaonan Wang a, Han Yang a, Xintong Geng a, Guoli Xu , Mickey Samaloa, MinaSakinatia, Danqun Huo b,* .Changjun Hou a,*

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a

Key Laboratory for Biorheological Science and Technology of Ministry of

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Education, State and Local Joint Engineering Laboratory for Vascular Implants,

Bioengineering College of Chongqing University, Chongqing 400044, PR China b

Liquor Making Biology Technology and Application of Key Laboratory of Sichuan

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Province, College of Bioengineering, Sichuan University of Science and Engineering,

Those authors contribute equally to this work.

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Zigong, 643000, PR China

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* Corresponding author. Tel.: +86 23 6511 2673; fax: +86 23 6510 2507.

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E-mail addresses: [email protected] (C. Hou)



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[email protected] (D. Huo)

Highlights

1. AChE-CS/3DG-CuO NFs could greatly amplify the electrochemical signal, and its good biological activity and high

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specific surface all provided favorable conditions for the detection of organophosphate pesticides (OPs).



2. The modified electrochemical biosensor exhibited a wide linear relationship to malathion ranging from 1 ppt to 15.555 ppb (3 pM-46.665 nM) and a low detection limit of 0.31 ppt (0.92 pM).



3. The recovery rates of water samples were in the range from 94% to 106%, which indicated that the developed biosensor

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had great potential application for pesticides detection.

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Abstract: In the present study, we developed a highly sensitivity electrochemical acetylcholinesterase (AChE, E.C.3.1.1.7) biosensor for organophosphorous pesticides (OPs) detection on the basis of three dimensional graphene-copper oxide nanoflowers nanocomposites (3DG-CuO NFs). The 3DG-CuO NFs nanocomposites with network-

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like structure not only increase the effective specific surface area, but also provide a favorable microenvironment for AChE loading, which could improve the biosensor

performance. The electrochemical performance of the AChE-CS/3DG-CuO NFs/GCE

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biosensor was thoroughly investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), amperometry (i–t) and square wave voltammetry (SWV). Under the optimal detection conditions, the AChE-CS/3DG-CuO NFs/GCE

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biosensor exhibits advantages such as a wide linear relationship to malathion ranging

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from 1 ppt to 15.555 ppb (3 pM-46.665 nM). The theoretical detection limit was

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calculated to be 0.31 ppt (0.92 pM) with good selectivity and ideal stability. Most

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importantly, satisfactory recoveries were achieved in real samples analysis, indicating that our developed biosensor has great potential to be an effective platform for

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pesticides detection.

Keywords: Three-dimensional graphene; CuO nanoflowers; Pesticides;

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Acetylcholinesterase

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1. Introduction Organophosphate pesticides (OPs), which occur in a diverse range of forms, set on a vital position in agriculture development worldwide as efficient and multipurpose pesticides [1]. Due to its low cost and high efficiency for eliminating pests and plant diseases, OPs are most widely used in practical agricultural production

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[2]. However, extensive use of OPs causes a series of problem in food safety and environment, such as OPs accumulation and residues [3-5]. Several methods has been

developed for rapid and reliable determination of OPs, such as liquid chromatography

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[6], gas chromatography [7], ultraviolet spectroscopy [8], fluorimetry [9] and

electrochemistry [10, 11]. Traditional analytical methods, such as liquid or gas chromatography suffer some limitations such as requirement of professional

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operation, time-consuming procedures, complicated sample pretreatment and costly

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[9], even though it has a good accuracy. Electrochemical sensors have acquired

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increasing attention for its simplicity, sensitivity, rapid response and great potential in

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miniaturization and instrumentation [11-13]. Enzymatic biosensor is a kind of outstanding electrochemical sensors, for which have both aforementioned advantages

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[14, 15]. Acetylcholinesterase (AChE) biosensors are the most commonly used for pesticide residues analysis in environmental monitoring, quality control, and food

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safety, due to their ultra-sensitivity, disposability, and provide rapid results [16, 17]. However, as a kind macromolecular protein, enzyme molecules always show poor

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conductivity, thus, various nanomaterials have drawn into biosensors fabrication to improve the conductivity, sensitivity, stability, and biosensor performance. Recently, nanomaterial/nanocomposites including 3D graphene [18, 19], carbon

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nanotube [18, 20, 21], [email protected] nanorods [22], NiO nanoparticles [23], TiO2 nanocomposites [24], CuO nanowires [25, 26] etc, have been widely employed in biosensor for better performance. Among those materials, graphene possesses many unique physical and chemical properties, such as favorable mechanical strength, high conductivity, good bio-compatibility [27, 28] etc. Furthermore, three-dimensional graphene (3DG), greatly updated and enhanced from typical monolayer graphene, has 3

high porosity and hydrophobicity owing to its porous network structure [29, 30]. Moreover, it also possesses a high specific surface area and supplies multiple active sites, which both benefits to further improve enzymes loading and incubation [31, 32]. Cu has excellent catalytic and electrical properties, which widely used in batteries, sensors and solar cells, especially in electrochemical biosensors. Copper oxide (CuO), a nontoxic material for p-type semiconductors, have shown great potential in wide

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applications (such as sensors and supercapacitors) due to its low cost, easy synthesis, and outstanding conductivity [33-35]. According to previous studies, Cu or Cu-

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containing compounds have a good affinity with thio-compounds, and thus have a potential to be exploited in OPs detection [36].

In this work, we constructed a new biosensor based on AChE-CS/3DG-CuO

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NFs/GCE to achieve sensitive and selective detection of OPs (Scheme 1). The

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nanocomposites of 3DG and CuO NFs can form network-like structure, which could

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increase the effective specific surface area and provide a favorable micro-environment

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for AChE loading. Chitosan (CS), a great biocompatible, film forming ability, nontoxicity and positively charged polymer, could be further employed to entangle with

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AChE and 3DG-CuO NFs nanocomposites [37, 38]. Under optimal conditions, the proposed biosensor exhibited excellent performance for monitoring malathion with a

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low detection limit and high sensitivity. The successful application for the detection of malathion in water samples further demonstrated its applicability for on-site

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detection of OPs in real samples.

2. Experiments

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2.1. Materials and reagents Acetylthiocholine chloride (ATCl) and acetylcholinesterase (AChE)

(E.C.3.1.1.7, 200 U/mg from electric eel) were obtained from Sigma-Aldrich. Malathion and other pesticides (methyl parathion (MP), deltamethrin (DM)) were provided by Chongqing Entry Exit Inspection and Quarantine Bureau. Graphene oxide (GO) was purchased from Nanjing Xianfeng Nanomaterials Co. Chitosan (CS, 4

AR) was acquired from Sinopharm Chemical Reagent Co., Ltd. The chitosan were dissolved in 50 mM acetic acid to obtain a 0.2 % CS solution. Phosphate buffered saline (PBS) with concentration of 0.1 M at pH 7.4 was freshly prepared. Other reagents were analytically pure and all the solutions were prepared with deionized water (DI) (18.2 M Ω·cm) by a Millipore Direct-Q Water system throughout the

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experiments. 2.2. Apparatus

Three dimensional graphene (3DG), copper oxide nanoflowers (CuO NFs) and

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3DG-CuO NFs nanocomposites were characterized by field emission scanning

electron microscope (FESEM, Nova 400, FEI corp.). Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), amperometry (i–t) and square wave

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voltammetry (SWV) were carried out to using a CHI 760E electrochemical

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workstation (Shanghai Chenhua Instruments Co. Ltd, China) with a conventional

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three-electrode system, using a platinum wire counter electrode, Ag/AgCl (3 M KCl)

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references electrode and modified GCE as working electrode. 2.3. Preparation of 3DG and CuO NFs

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According to previous researches [39, 40], the aqueous GO dispersion prepared at concentration of 2.5 mg/ml was placed in an autoclave and heated at 180°C for 12h.

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Over the sufficient reaction, the 3DG hydrogel was freeze dried and then made into 2 mg/ml dispersion with DI water. The synthesis of CuO NFs were following the

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method described by Sun with a slightly modification [34]. The black sediment (CuO NFs) was suspended at 2 mg/ml in anhydrous ethanol. Two aqueous dispersion were mixed with an appropriate volume ratio (ratio 2:1 v/v), and then ultrasonically stirred

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for 30 min. The 3DG-CuO NFs dispersion were prepared for further use. 2.4. Fabrication of AChE-CS/3DG-CuONFs modified GCE The glassy carbon electrode (GCE, 3mm in diameter) were polished with 1 and 0.05 μm alumina slurries, sonicated the GCE in acetone, ethanol and DI water for 30 s until the mirror-like surface were obtained and the electrode were dried with purged nitrogen. The polished electrodes were scanned electrochemically by cyclic 5

voltammetry (CV) in 0.1 M KCl solution containing 5 mM [Fe(CN)6]3-/4- (Range from -0.2 to +0.6 V, scan rate of 50 mV/s). The peak potential differences lest than 95 mV were considered as qualified. Then 6 μL 3DG-CuO NFs dispersion were dropped onto the surface of the qualified GCE and were dried at the room temperature for several hours. Then, 6 μL

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of a mixture containing AChE and Chitosan (AChE:CS ratio 2:1 v/v , 0.2% wt CS) was further immobilized on 3DG-CuO NFs/GCE and incubated at 4°C for 10 h. Throughout, the AChE-CS had been immobilized. The AChE-CS/3DG-CuO

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NFs/GCE biosensor was accomplished and stored at 4°C for further use. 2.5. Electrochemical measurements

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Malathion was chosen as the pesticide model of this study, for it was widely used in the modern agriculture and also an outstanding and sensitive AChE inhibitor. The

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prepared AChE-CS/3DG-CuO NFs/GCE biosensor was dipped in 0.1M PBS

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(pH=7.4) containing 0.5 mM ATCl and square wave voltammetry (SWV, from 0.2 to

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1 V, scan rate of 50 mV/s) was done and the original SWV images were recorded. Then, the typical electrode were immerged in a series of concentration gradient of

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malathion about 10 min incubation for each concentration. Finally, SWV measurements were carried out for a second time in the same conditions. The

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inhibition of malathion were calculated as I 0 -Ii

I (%) =

×1 0 0

I0

In this formula, I0 and Ii represent the peak currents of 0.5 mM ATCl before and

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after the malathion incubation, respectively.

3. Results and discussion 3.1. Characterization of the 3DG/CuO NFs Fig.1 shows field emission scanning electron microscope (FESEM) image was done to investigate the morphology of these materials. SEM image of threedimensional graphene hydrogel (3DG) shows that a three-dimensional porous fluffy 6

network-like structure was emerged with a large number of pleated surface area (Fig. 1A). The SEM image of CuO NFs shows that the shape of the CuO NFs were round layered flowers (Fig. 1B) which was conducive to material adsorption and enhancement of the biosensor. Fig. 1C and 1D were the characterization of 3DG-CuO NFs nanocomposites, it could clearly see that the flower-like CuO NFs had uniformly dispersed on the 3DG lamellae. The composite structure provided a favorable

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biocompatibility environment for immobilization of AChE and strongly promoted the transfer rates during the electrochemical reaction because of its great conductivity.

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3.2. Electrochemical behavior of the biosensor

Cyclic voltammetry (CV) is generally used to study the electrochemical

properties of biosensor in solution. As shown in Fig. 2A, bare electrode (curve a) and

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respective modified electrodes were investigated as redox probe in 0.1M pH 7.4 PBS

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buffer containing 5 mM [Fe(CN) 6] 3-/ 4-. It can be distinctly seen that the current

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signal of the electrodes modified with CuO NFs, 3DG and 3DG-CuO NFs (curve b-d)

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increased sequentially, which proved that the 3DG-CuO NFs nanocomposite electrode has excellent conductivity and adsorption, more importantly, it can effectively

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promote electron transfer rates. The spacious surface area framework formed by the hybrid composite system provides a large number of active sites for AChE loading.

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The peak current (Fig. 2A, curve e) decreased obviously when AChE molecules were immobilized on the 3DG-CuO NFs modified GCE, for the protein is non-conductive,

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thus the electron transfer rates of the system was decreased. Electrochemical impedance spectroscopy (EIS) is used to study the interfacial

properties of different materials modified electrodes, providing more theoretical basis

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for the study of sensor. The Nyquist plot is the most common output of impedance data and suitable for evaluating the impedance changes in sensing systems [41, 42]. Various modified electrodes were subjected to EIS in similar condition (Fig. 2B). With the employment of modified materials, it can be observed that the 3DG-CuO NFs/GCE shows the lowest impedance (Fig. 2B, curve d). The obvious decrease in Rct value proved that the 3DG-CuO NFs/GCE has a great conductivity, which is 7

benefit for electron transfer. On the other hand, the addition of AChE toward the modified electrode caused Rct increases significantly (Fig. 2B, curve e). This phenomenon can be associated to the thickness of the interface after AChE immobilization on the electrode, for enzyme was non-conductivity, thus hindered the electron transfer. However, it also indicated that the AChE has immobilized on the

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modified electrode successfully. As shown in Fig. 3A, peaks were observed at AChE-CS/3DG-CuONFs/GCE

(curve b) in the presence of 0.5 mM ATCl, mostly because of the hydrolysis towards

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ATCl. With the increasing concentrations of ATCl, the peak current at ca. +0.6 V

(Fig. 3B) rose distinctly and the peak reached the maximum at 0.5 mM ATCl, which means 0.5 mM is a probably saturated concentration. The maximum current (Fig. 3B,

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curve d) was much higher than peak A (Fig. 3A, curve b) due to the fabricated of

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3DG-CuO NFs, signifying that this sensor has excellent electrochemical performance.

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3.3. Optimization of experimental conditions

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To further optimize the fabricated electrochemical biosensor for the quantitative malathion assay, the pH of PBS, the incubation time in the pesticide and the amount

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of AChE were investigated by SWV measurement and the corresponding results are displayed in Figs.S1-S3. To optimize each parameter, all other parameters were

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operated under their optimal conditions.

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As shown in Fig S1, the effect of pH (from 6.0 to 8.5) on enzyme activity at the developed AChE-CS/3DG-CuONFs biosensors was researched. It can be seen that the maximum current obtained at pH 7.4, therefore it was selected as the optimum pH

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value in subsequent experiments. Besides, the incubation time can also influence the performance of the

constructed biosensor. The current showed significant decrease with increasing incubation time within 600 s and plateaued after 600 s (Fig. 4B), indicating that hybridization was complete within 10 min. Thus, 600 s was selected as the optimal incubation time. 8

The amount of AChE immobilized on the biosensor was another important aspect. As shown in Fig. S3, with the increase of AChE from 0.067 to 0.33 U, the current response of the biosensor to 0.5 mM ATCl increased initially and then reached a maximum at 0.2 U. It can be attributed to that a small quantity of AChE could lead to catalyze insufficiency of the biosensor, but further increasing the amount of AChE led to the increasing in mass transfer resistance and inaccessibility, which caused a

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slight decrease of the current signal. Thus, 0.2 U of AChE was employed in following experiments.

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3.4 Amperometric response toward ATCl

Amperometric response was chosen to further study the electrocatalytic

performance of proposed biosensor toward ATCl. The current-time curve of AChE-

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CS/3DG-CuO NFs/GCE with successive addition of ATCl into constantly stirred PBS

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(pH=7.4) at 0.6 V are demonstrated in Fig. 4A. With an increase in ATCl

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concentration from 0.5 μM to 100 μM, the current signal responded quickly and rose

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gradually. Therefore, the oxidation current and the concentration of ATCl showed a good liner correlation. The as calculated equation y=0.02045x+2.68841 (R2=0.996)

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(Fig. 4B) shows that the sensor has a great sensitivity towards ATCl and benefits further detection of OPs.

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3.5. Pesticide determination

Square wave voltammetry (SWV) was applied for further electrochemical

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detection owing to its great sensitivity. Fig. 5A displayed the SWV of the AChECS/3DG-CuO NFs/GCE in 0.1 M PBS solution (pH 7.4) after incubation with various malathion concentrations. The peak currents decreased sharply with the increasing

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concentration of malathion due to the inhibitory effect on AChE. The calibration curve of inhibition (ΔI) versus logarithmic of pesticide concentration (log C) is shown in Fig. 5B. The linear equation is ΔI = 2.793 log C + 10.536 (R2 = 0.991, n=3) from 1 ppt to 15.555 ppb (3 pM to 46.665 nM). The limit of malathion detection was estimated to be 0.31 ppt (S/N=3), which is much lower than previous studies and others works done using various nanomaterials or graphene nanocomposite 9

(summarized in Table 1). Such low detection of limit and great biosensing performance might be attributed to the 3DG-CuO NFs nanocomposite with excellent conductivity and large specific surface area can accelerate the electron transfer, and it allow immobilization of AChE molecules. 3.6. Interference, reproducibility and stability studies

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To further identify the anti-interference and selectivity of the AChE-CS/3DGCuO NFs/GCE biosensor, interference studies were done with the presence of several common interfering species, containing different pesticides, glucose, metal ions and

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inorganic salts. The SWV response was normalized on basis of optimized conditions for 1 ppb of malathion. Compared with the standard signal, there were no significant

changes in the signals among the presence of 1.0 mM glucose, NO3-, PO43-, SO42- and

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Cu2+. However, in the coexistence of methyl parathion (MP), deltamethrin (DM) and

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Pb2+, the current signal was interfered to certain degrees (66.65%, 83.12% and

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78.96%, respectively), which might be explained to the fact that the inhibition of

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AChE activity by pesticides and toxic compounds lacks of specificity. In general, the AChE-CS/3DG-CuO NFs/GCE/GCE biosensor resisted interference well and

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exhibited acceptable selectivity towards pesticides used in the study. Reproducibility and stability are also important factors in the practical use of

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OPs biosensors. The reproducibility with a relative standard deviation (RSD) of 4.7% was evaluated by five different AChE-CS/3DG-CuO NFs/GCE containing 0.5 mM

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ATCl, indicating an acceptable reproducibility of the proposed biosensor. Additionally, the stability of developed AChE biosensor was assessed by long-term monitoring the electrochemical signal at specified conditions in 0.1 M PBS solution

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(pH 7.4) containing 0.5 mM ATCl with the presence of malathion, the current responses of the biosensor were stored at 4°C and tested for 20 consecutive days. After 20 days, the response current of the biosensor still retain 93.70% of the initial current signal, demonstrating excellent storage stability of the biosensor.

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3.7. Real sample analysis To further investigate the applicability of the fabricated biosensor, real water samples were collected from Yangtze River (part from Chongqing, China) and Minzhu Lake (Chongqing University, China). They were first filtered through a welldefined 0.2 μm PVDF filter to remove any large particles, then centrifuged to get two real water samples and the pH and ionic strength were adjusted to match with the

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buffer solution used in previous sections. Two samples were used to detect malathion using AChE-CS/3DG-CuO NFs/GCE biosensor by standard addition method. As

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listed in Table 2, the recoveries were in the range of 94%-106%, indicating that our biosensor has a great performance in OPs detection in real samples analysis.

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4. Conclusion

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In summary, an efficient electrochemical AChE biosensor based on 3DG-CuO

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NFs nanocomposites for OPs detection were successfully constructed. The fabricated AChE biosensor was also systematically optimized with the operating pH of the PBS,

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the incubation time in the pesticide and the amount of AChE loading. The AChE-

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CS/3DG-CuO NFs/GCE biosensor exhibited excellent performance toward malathion with large linear range, high sensitivity, outstanding reproducibility and stability. To demonstrate the practical application, the detection of spiked malathion in Yangtze

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River and Minzhu Lake samples were successfully tested on the designed biosensor. These results indicate that the proposed sensor is expected to be a new efficient

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approach for on-site detection of organophosphorus pesticides with high performance.

Acknowledgement

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Author Biographies

Jing Bao is presently a Ph.D. student in College of Bioengineering at Chongqing University, People’s Republic of China. Her research interests include the development of new nanomaterials for application in optoelectronic devices and analysis. Ting Huang is currently studying for bachelor's degree in Bioengineering College of Chongqiong University. Her research interests lie in sensors and technology of biochemical sensors detections. 11

Changjun Hou completed his Ph.D. in biomedical engineering in 2004 at Chongqing University. After a period of research at the University of Illinois at Urbana-Champaign as a visiting scholar, he returned to China and got tenure at the college of bioengineering in Chongqing University. In 2010, he became a fullprofessor in biomedical engineering. His scientific interests rest on the design and construction of biochemical sensing systems based on porphyrins and other sensitive materials. Danqun Huo received her Ph.D. degree in biological medical engineering from

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Chongqing University in 2004 and had once been visited Australia, America, and Italy. She is currently Distinguished Professor of Sichuan University of science and

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engineering and professor of biomedical science of Chongqing University. Her current

research interests include the development of chemical sensors and sensor array, electrochemistry and analytical microsystems.

This work was supported by the National Natural Science Foundation of China (NO.

81772290 and 81271930), Graduate Scientific Research and Innovation Project of Chongqing

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(CYB17037), Chongqing University innovation training program for College Students

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(201710611100), the workstation in Sichuan Province GY2015-01 and sharing fund of

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Chongqing university's large equipment.

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of acetylcholinesterase on NiO nanoparticles-carboxylic graphene-nafion modified electrode for

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chemical and biological sensors, Sensors and Actuators B: Chemical, 231(2016) 324-40.

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microspheres for high-efficiency adsorption, Journal of Materials Science, 52(2017) 13930-9.

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inspired layered materials of 3D graphene network/calcium carbonate, Journal of Wuhan

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prepare graphene foam-like three-dimensional porous carbon/Ni nanoparticles for glucose

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Determination

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Functionalized Magnetic Iron Nanoparticles, Biosensors, 8(2018).

of

Malathion

Using

Acetylcholinesterase

18

Immobilized

on

Chitosan-

132

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assembly

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organophosphates pesticide, Sensors and Actuators B: Chemical, 146(2010) 337-41.

β-cyclodextrins

onto

multiwall

carbon

nanotubes

for

detection

of

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of

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Figures and table captions Scheme 1. Schematic illustration of electrochemical AChE-CS/3DG-CuO NFs/GCE

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biosensor

137

Fig.1. FESEM images of 3DG (A), CuO NFs (B), 3DG-CuO NFs (C, 10 μm) and

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3DG-CuO NFs (D, 2 μm)

139

Fig.2. CVs (A) and EISs (B) of different modified electrodes in 0.1 M pH 7.4 PBS

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containing 5 mM [Fe(CN)6]3-/4-, (a) bare GCE, (b) CuO NFs/GCE, (c) 3DG/GCE, (d)

141

3DG-CuO NFs//GCE, (e) AChE-CS/3DG-CuO NFs/GCE.

142

Fig.3. (A) CVs of AChE-CS/GCE in the absence and presence of 0.5 mM ATCl in

143

0.1 M pH 7.0 PBS; (B) CVs of AChE-CS/3DG-CuO NFs/GCE in 0.1 M pH 7.4 PBS

144

containing different concentrations of ATCl, (a) 0 mM, (b) 0.1 mM, (c) 0.2 mM, (d)

145

0.5 mM.

146

Fig.4. (A) Amperometric responses for the sensor on successive addition of ATCl

147

(0.5, 1, 5, 10, 50 and 100 μM) to 0.1 M pH 7.4 PBS, constantly stirring. Operating

148

potential: 0.6 V. (B) The calibration curve for ATCl determination.

149

Fig.5. (A) SWV responses of AChE-CS/3DG-CuO NFs/GCE/GCE in 0.1 M pH 7.4

150

PBS solution with 0.5 mM ATCl after incubation with malathion for 5 min (a-k).

151

Malathion concentration (a)-(h): 0 ppt, 1 ppt, 4 ppt, 10 ppt, 40 ppt, 0.1 ppb, 0.4 ppb, 1

152

ppb, 4 ppb, 10 ppb, 40 ppb. (B) The corresponding calibration plot for malathion

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determination.

Fig.6 Amperometric response of AChE-CS/3DG-CuONFs/GCE/GCE in 0.1 M pH 7.4 PBS solution containing 1 ppb of malathion (mal) and 0.5 mM ATCl (a) with coexistence of 1 ppb

A

155

CC E

153

IP T

135

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methyl parathion (MP) (b), 1 ppb Deltamethrin (c) 1.0 mM glucose (d), 1.0 mM NO3- (e), 1.0

157

mM PO43- (f), 1.0 mM SO42- (g), 1.0 mM Pb2+ (h) and 1.0 mM Cu2+ (i), respectively. Error

158

bars: SD, n=3.

159

Table.1. Recovery studies of spiked malathion in Yangtze River and Minzhu Lake (n=3)

160

20

Table.2. Comparisons of the proposed AChE biosensor with other biosensors for the

162

detection of malathion.

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A ED

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Scheme 1

22

A ED

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M

165

Figure 1

23

A ED

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CC E

168

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167

Figure 2

24

A ED

PT

CC E

170

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169

Figure 3

25

A ED

PT

CC E

172

IP T

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U

N

A

M

171

Figure 4

26

Figure 5

IP T

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SC R

174

A

CC E

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ED

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N

U

175

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Figure 6

28

Table 1 AChE biosensors

Linear range

Detection limit

References

AChE/Chit-PB-MWNTs-HGNs/Au

0.05-75 nM

0.05 nM

[37]

AChE&ChOx/Pt-Au/MWCNT/GCE

0.1–50 nM

0.16nM

[43]

AChE-Cs/Pb-Cu NWs/GCE

15-3000 pM and 1500-

4.5 pM

[44]

0.3 nM

[45]

AChE-CS/Fe3O4/SPCE

0.5-20 nM

IP T

9000 nM *

0.01-10.0 μM

2 nM

[46]

AChE-PAn-PPy-MWCNTs/GCE

0.03-1.5 and 3-75 mM

3 nM

[20]

AChE-CS/3DG-CuO NFs/GCE

3 pM-46.665 nM

0.93 pM

This work

SC R

AChE-MWCNTs-β-CD-CHIT/GCE

A

CC E

PT

ED

M

A

N

U

*SPCE: Screen-Printed Carbon Electrode

29

Table 2 Recovery (%)

RSD (%)

1.00

1.064

106.4

2.37

10.00

9.433

94.33

1.89

20.00

20.518

102.59

4.15

1.00

1.036

103.4

10.00

10.683

106.83

20.00

19.098

95.49

A

CC E

PT

ED

M

A

N

U

Minzhu Lake

Found (ppb)

30

IP T

Yangtze River

Spiked (ppb)

SC R

Sample

3.29 4.17 2.04