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Fluorescence determination of omethoate based on dual-strategy for improving sensitivity Cuiping Zhang, Bixia Lin, Yujuan Cao, Manli Guo, and Ying Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00166 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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Title: Fluorescence determination of omethoate based on dual-strategy for improving sensitivity

Full Names of the Authors: Cuiping Zhang, Bixia Lin, Yujuan Cao, Manli Guo, Ying Yu*

Affiliations: School of Chemistry and Environment, Guangzhou Key Laboratory of Analytical Chemistry for Biomedicine, South China Normal University, Guangzhou 510006, P. R. China.

Corresponding author: : Prof. Ying Yu Tel: +86-20-39310382 Fax: +86-20-39310187 E-mail: [email protected]

Present/permanent address: : No. 378 Waihuan West Road, University City, Guangzhou, Guangdong, P. R. China, 510006

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Fluorescence determination of omethoate based on dual-strategy for improving sensitivity 1

Cuiping Zhang, Bixia Lin, Yujuan Cao, Manli Guo, Ying Yu*

2 3

School of Chemistry and Environment, Guangzhou Key Laboratory of Analytical Chemistry for Biomedicine, South China Normal University, Guangzhou 510006, P. R. China. 4 5

Abstract

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Omethoate is a frequently-used organophosphorus pesticide, the establishment of a sensitive,

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selective and simple method to determine omethoate is very important for food safety. In this

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paper, dual-strategy was applied to improve the detection sensitivity of omethoate. The first

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strategy, graphene quantum dots (GQDs) were doped with nitrogen to increase the fluorescence

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quantum yield to 30%. By coupling N-GQDs with omethoate aptamer, N-GQDs-aptamer probe

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was synthesized. The fluorescence of N-GQDs-aptamer probe was turned off by graphene oxide

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(GO), but recovered by omethoate. Based on this principle, the fluorescence method for detecting

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omethoate was established with detection limit of 0.041 nM. To further improve the detection

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sensitivity, fluorescence polarization analysis method was applied as another strategy based on the

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polarization signal of GQDs. The detection limit was decreased to 0.029 pM by using fluorescence

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polarization method. The detection limits in this paper were lower than those in other reports. The

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imaging of omethoate on plant leaves shown the probe could be used for visual semi quantitative

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determination of omethoate.

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Keywords: dual-strategy; nitrogen-doped graphene quantum dots; omethoate; fluorescence

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method; fluorescence polarization

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1. Introduction

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Organophosphorus pesticide is one of the most commonly used pesticides. The over-use of

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organophosphorus pesticide has threat to human health and harmful effects on the ecology, so it is

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very important to develop an effective detection method. Omethoate is an organophosphorus

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pesticide with the simplest chemical structure. Due to the lack of sensitive luminophore or

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electrochemical active groups, it is difficult to determine omethoate directly by using conventional

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optical analysis or electrochemical method. Until now it is still a challenge to establish a detection

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method with good selectivity, high sensitivity and convenience for the determination of low

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content of omethoate in complex samples.

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Most of the reported detection methods for omethoate were gas chromatography1, liquid

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chromatography2, gas chromatography/mass spectrometry3,4, capillary electrophoresis method5.

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These methods are often combined with complex separation process such as magnetic separation1,

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solid phase micro extraction6,7 and molecular imprinting8. Other methods such as electrochemical

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analysis9,10, Raman spectroscopy11-13, optical analysis method14-18 were also reported for

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determining omethoate. In the literature about optical analysis, Zhang14 used molecularly

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imprinted polymer separation and Sun15 used flow injection separation combined with

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chemiluminescence analysis to establish the omethoate determination method. Pang16 utilized the

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oxidation of R-DmAChE and indoxyl acetate as substratechans to produce blue products. When

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organic phosphorus pesticides or carbamate pesticides were present, the R-DmAChE enzyme was

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consumed and the blue product was reduced. Thus the rapid visualization colorimetric detection of

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organic phosphorus pesticides and carbamate pesticide were realized. Fluorescence method was

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popular in the optical analysis. Ji17 reported that the molecular beacon was modified on the surface

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of gold nanoparticle, and their fluorescence was quenched by gold nanoparticle. When ss-DNA

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was present and bound to the small peptide of the molecular beacon, the fluorescent group in the

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molecular beacon was far away from the gold nanoparticle, and the fluorescence was recovered.

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When the analyte and the ss-DNA were coexistent, the analyte combined to ss-DNA, so the

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fluorescence intensity of the molecular beacon was recovered partly. Based on the above principle,

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isocarbophos, profenofos, phorate and omethoate were detected. Liu18 reported the fluorescence of

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hairpin molecular beacon was quenched by organophosphorus pesticide and ss-DNA binding, and

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recovered by hairpin molecular beacon and ss-DNA combination. Thus the determination of four 3

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pesticides including omethoate was realized. Although there are some reports on optical analysis

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method for omethoate determination, the sensitivity, selectivity, convenience still need to be

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improved.

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In recent years, optical sensor has attracted the attention of researchers. Optical sensor

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consists of two parts, the optical signal component and the recognition component. To construct

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optical signal components, in addition to the traditional colored materials and fluorescent dyes, the

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nano materials such as Cd system quantum dots19,20, manganese doped ZnS21, MoS222, carbon

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dots23,24, silicon dots25 and gold nanoparticle26,27 are used widely. To construct recognition

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components, small molecules28,29, single-stranded DNA22,30 and antibodies24 can be used.

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Fluorescent sensors are the most common optical sensors. They have been constructed

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successfully to detect all kinds of targets, for example, our group24 had designed fluorescence

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probe for glyphosate by modifying carbon dots with glyphosate antibodies. After the probe

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combined to glyphosate, the excess probe was removed with glyphosate magnetic beads. Thus a

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sensitive detection method for glyphosate was established. Nowadays, fluorescence polarization

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sensors which is based on the mechanism of the depolarisation of linearly polarised light by

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fluorophore molecules with anisotropy is also used for the targets detection31. Layered

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nanomaterial such as graphene oxide is reported to be suitable for constructing fluorescence

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polarization sensors32.

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In order to establish a sensitive, convenient and rapid optical analysis method for omethoate,

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in this paper, luminescent probe (N-GQDs-aptamer) with recognition function was synthesized

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and the double strategies of doping nitrogen and fluorescence polarization were used for

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improving the detection sensitivity. In the first strategy, the quantum yield of N-GQDs was

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increased 30% by doping nitrogen in the synthesis of graphene quantum dots. The fluorescence of

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N-GQDs-aptamer probe was turned off by graphene oxide, but recovered by omethoate. Based on

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this "off-on" sensor, the fluorescence method for omethoate was established with detection limit of

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0.041 nM. Because the GQDs had fluorescence polarization signal, fluorescence polarization

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analysis method was also established with low detection limit of 0.029 pM. The detection limit of

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fluorescence method was about 1000 times of fluorescence polarization method. This meant the

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fluorescence polarization method could be applied as the second strategy to improve the detection

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sensitivity for omethoate. By applying the dual-strategy for improving sensitivity, the sensitivity 4

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of detection methods established in this paper was superior to the other methods in previous

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reports. Furthermore, the probe could be directly used for imaging of residual omethoate on leaves

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surface, the method was simple, quick and had the potential for on-site detection. The detection

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mechanism was shown in Scheme.

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Scheme

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2. Materials and methods

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2.1 Materials and Instruments

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Graphene oxide with thickness of 0.8-1.2 nm was purchased from Nanjing xfnano Mstar

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Technology Ltd. Urea was purchased from Tianjin Damao Chemical Reagent Factory.

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Carbodiimide hydrochloride (EDC·HCl, 98.5%), N-hydroxysuccinimide (NHS, 98%), omethoate

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(99%), NaOH, Na2HPO4·12H2O and NaH2PO4·2H2O were purchased from Aladdin Chemical

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Company. Aptamer (5′-NH2-AAGCT TTTTT GACTG ACTGC AGCGA TTCTT GATCG

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CCACG GTCTG GAAAA AGAG-3′) was purchased from Shanghai Sangon Biological

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Engineering Co. Ltd. All chemicals used were of analytic grade or of the highest purity available.

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Double-distilled water was used throughout the experiments.

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Fluorescence spectra were obtained using an F-4600 fluorometer with polarizer (Hitachi,

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Japan) under voltage of 400 V, slit width of 5 nm and scanning speed of 300 nm/min. UV-vis

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absorption spectra were measured with a 2700 UV-vis spectrophotometer (Shimadzu, Japan).

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Transmission electron micrographs were carried out on 2100 HR microscope (Hitachi).

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Fluorescence images were performed with an OLYMPUS IX73 biological fluorescence

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microscope (Olympus Co. Ltd).

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2.2 Synthesis of N-GQDs

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N-GQDs were prepared using a modified method33. Briefly, 0.0251 g graphene oxide, 40 ml

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distilled water were added into 100 ml beaker and ultrasonic for 60 min at 180 w, 59 kHz. After

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the mixture was disperse evenly, 400 µL of 0.625 mg/ml urea solution was added. The pH was

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adjusted to 7 with 0.1 M NaOH solution, the mixture was transferred to Teflon autoclave and

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reacted at 200 °C for 12 h. After the reaction was completed, the reaction mixture was cooled to

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room temperature and filtered through a 0.22 µM microfiltration membrane to remove black

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insoluble matter. The concentration of brown-yellow filtrate calculated by graphene oxide was

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0.625 mg/ml. The filtrate was dialyzed with 1000 Da dialysis bags, 500 ml secondary distilled 5

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water and the water was changed every 30 min until the conductivity of the dialyzate near that of

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distilled water. Finally the dialyzate was collected and stored at 4 °C.

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2.3 Synthesis of N-GQDs-aptamer probe

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N-GQDs-aptamer was prepared using a modified method33. Briefly, 5.0 ml N-GQDs, 5 ml

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PBS at pH 7.0 and 1.2 ml of 8 mM/2 mM EDC/NHS mixed solution were sequentially added to

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the flask and stirred for 30 min. Then 19 µL of 100 µM omethoate aptamer solution was slowly

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added and stirred for 18 h. A clear yellow solution of N-GQDs-aptamer was obtained, and the

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concentration of N-GQDs-aptamer calculated by graphene oxide was 0.279 mg/ml.

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2.4 Determination of omethoate in actual samples

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The working curve for determination of omethoate: 0.5 ml omethoate standard solution at

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different concentrations, 1.0 ml GO...N-GQDs-aptamer solution, 3.5 ml PBS solution at pH 6.5

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were added to 5 ml colorimetric tubes and diluted to a constant volume of 5.0 ml. After incubating

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at room temperature for 45 min, the fluorescence intensity of mixture was measured at λex/λem of

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310/413 nm. The working curve was plotted between the fluorescence intensities and omethoate

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concentrations. The parallel polarization intensity (i// and I//) and vertical polarization intensity (i⊥

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and I⊥) of above solutions were also measured at λex/λem of 310/413 nm. The polarization intensity

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(P) was calculated according to the formula P =

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for fluorescence polarization method was also plotted between the polarization intensities and

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omethoate concentrations.

I// − G × I ⊥ and G = i ⊥ , and the working curve I// + G × I ⊥ i//

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Preparation of actual samples: The water samples were originated from the Pearl River,

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filtered to remove insolubles and stored at 4 °C. 2.0 g cabbage sample was crushed with a mortar

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and transferred to a beaker. 50 ml of the double-distilled water was added, and the mixture was

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extracted with ultrasonic for 20 min. The extractant was filtered, transferred to the flask and

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diluted to constant volume of 100 ml and stored at 4 °C.

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Measurement of actual samples: 0.5 ml of the prepared sample solution, 1 ml of 162 nM

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GO...N-GQDs-aptamer probe, 3.5 ml PBS at pH 6.5 were transferred into 5 ml colorimetric tube.

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After diluting to 5 ml, the sample solutions were measured at λex/λem of 310/413 nm, the amount

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of omethoate in the samples was calculated according to the working curves. The standard

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addition method was used to determine the recovery rate. Briefly, in a series of 5 ml colorimetric 6

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tubes, 0.5 ml of prepared sample solution, different concentrations of omethoate were added, the

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final spiked concentrations of omethoate were 0.10 pM, 0.050 nM and 4.0 nM. The spiked

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samples were measured at λex /λem of 310/413 nm, and the recovery rates for each method were

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calculated.

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2.5 Imaging of omethoate in plants

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Cabbage leaves were dripped with omethoate pesticide at different concentrations of 20, 5,

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0.1 µg/L, followed by adding with 162 nM of GO...N-GQDs-aptamer. After laying at room

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temperature for 45 min, the cabbage leaves were observed under IX73 fluorescent biological

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microscope with 40X objective lens. The blank and leaf with only omethoate or

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GO...N-GQDs-aptamer were set as control groups.

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3. Results and discussion

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3.1 Synthesis and Characterization

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The probe designed was composed of two parts, the optical signal component and the

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recognition component. N-GQDs with good biocompatibility were chosen as optical signal

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component. Omethoate aptamer was chosen as the recognition component and assembled on the

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surface of N-GQDs by coupling agents EDC and NHS.

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GQDs emitted blue fluorescence at 410 nm with excitation at 320 nm. In order to increase the

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fluorescence intensity, GQDs was doped with nitrogen. The effects of doping amount, pH value,

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reaction time and reaction temperature on the fluorescence intensity of N-GQDs were investigated.

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The results in Figure 1A shown when the doping amount of urea was 1.0%, the reaction pH was

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7.0, the reaction time was 12 h and the reaction temperature was 200 °C, the strongest

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fluorescence intensity was achieved. After obtaining the N-GQDs with good fluorescence

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properties, N-GQDs were modified with omethoate aptamer to obtain the N-GQDs-aptamer

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probes. The effects of different conditions for the synthesis of N-GQDs-aptamer probe were also

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tested. Because the suitable temperature for aptamer was no more than 25 °C, the coupling

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reaction was carried out under 25 °C. The effects of other conditions were shown in Fig. 1B. The

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results shown the optimal conditions were the mass ratio of aptamer was 1.0%, the pH was 7 and

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the coupling reaction time was 18 h.

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Figure 1

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Fig. 2A shown the fluorescence spectra of GQDs, N-GQDs and N-GQDs-aptamer. Both the

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emission peaks of GQDs and N-GQDs were 423 nm, a new excitation peak at 260 nm could be

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observed in the excitation spectra of N-GQDs. This excitation peak could be ascribed to the

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formation of more complex energy level causing by nitrogen doped31. Furthermore, the

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fluorescence intensity of N-GQDs was increased about one time, and the quantum yield reached

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about 30% when using quinoline sulfate as reference substance. These meant the doped nitrogen

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greatly improved the fluorescence property of GQDs. N-GQDs shown strongest fluorescence

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signa at λex / λem = 320 nm / 423 nm, while the excitation and emission peaks of N-GQDs-aptamer

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were blue shifted to λex / λem =310 nm / 413 nm. And the fluorescence intensity of

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N-GQDs-aptamer probe was approximately doubled compared with that of N-GQDs. The blue

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shift of excitation and emission wavelength, the enhancement of fluorescence intensity proved that

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aptamer was successfully coupled to GQDs. The coupling was also further confirmed by the

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fluorescence polarization experiment. Fig. 2B showed that the polarization spectrum of N-GQDs

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had a significant peak at 360 nm, indicating N-GQDs were not dots but roundles. However, when

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N-GQDs were coupled with aptamer, the polarization peak at 360 nm disappeared. This was

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because the aptamer on N-GQDs decreased the directionality of N-GQDs-aptamer.

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The morphology and size of N-GQDs and N-GQDs-aptamer were characterized by

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transmission electron microscopy. The experimental results are shown in Fig. 2C. It could be seen

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that N-GQDs had good dispersibility, and the particle diameter was 5 nm. Because the layer

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thickness of graphene oxide was about 0.8 to 1.2 nm, it could be concluded that N-GQDs were

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roundles with diameter of about 5 nm and thickness of about 0.8~1.2 nm. No significant change in

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particle size and dispersibility was observed after the modification of omethoate aptamer. The

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hydrodynamic diameter and zeta potential of N-GQDs and N-GQDs-aptamer in Fig. S1 and S2

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also proved the successful conjugation of aptamer on N-GQDs.

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Figure 2

198 199

3.2 Construction of off-on sensor

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The prepared N-GQDs-aptamer probe had obvious fluorescence at λex / λem = 310/413 nm.

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After different carbon nanomaterials were added, the fluorescence was quenched diversely.

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However, some of the fluorescence could be recovered by omethoate added. As shown in Fig. 3A, 8

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the fluorescence of the probe was quenched by the same amount of carbon dots, carbon nanotube

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and graphene oxide to about 40%, 55% and 100%, respectively. When omethoate was added, only

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the fluorescence quenched by graphene oxide was almost fully recovered. Therefore, GO could be

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used as the quencher.

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The effects of the GO amount and the quenching time on the fluorescence intensity of

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N-GQDs-aptamer probe were further studied. The results in Fig. 3B1 and Fig. 3B2 shown the

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fluorescence intensity of the N-GQDs-aptamer probe decreased with increasing the amount of GO

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and quenching time. When the concentration of GO was 0.16 mg/ml and the quenching time was

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45 min, the fluorescence of the probe was almost completely quenched. The effect of GO on the

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fluorescenec intensity of N-GQDs was served as control group in Fig. 3B3. The results showed

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that both the fluorescence of N-GQDs-aptamer and N-GQDs could be quenched by GO. But the

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quenching effect on N-GQDs-aptamer was greater than that on N-GQDs. Figure 4A shown only

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the existence of GO could cause the obvious fluorescence quenching, and the absorption spectrum

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of GO was overlapped partly with the fluorescence spectrum of N-GQDs-aptamer. Thus, it was

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reasonable to consider that the fluorescence quenching of N-GQDs was due to the collision of the

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increased particles in the solution when GO was added. When N-GQDs were conjugated with

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omethoate aptamer, which got close to GO due to the strong π-π stacking interactions between

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aptamer over N-GQDs surface and GO, therefore, the probability of fluorescence resonance

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energy transfer between N-GQDs and GO increased, leading to the greater quenching effect. This

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was also confirmed by the TEM in Figure 4B. After adding GO to N-GQDs-aptamer,

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N-GQDs-aptamer could been found adhering onto the surface of GO sheet.

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Figure 3

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Figure 4

226 227

3.3 Sensitivity and selectivity of the probe

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Based on the fluorescence recovery of N-GQDs-aptamer by omethoate, a sensor for the

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determination of omethoate was constructed. The effects of buffer system and dosage, reaction

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time on fluorescence recovery were tested. The results in Figure S3 shown the optimal buffer was

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3.5 mL PBS with pH 6.5, the optimal time was 50 min. As illustrated in Figure S4, under the

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optimal conditions, the fluorescence intensity of probe was proportional to the concentration of 9

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omethoate in the range of 0.1 to 17 nM with the equation of △F = 56.42 + 73.32c (nM), the

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correlation coefficient R2 was 0.999, and the detection limit was 0.041 nM. Since

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N-GQDs-aptamer had polarization signal, polarization fluorescence could be utilized to measure

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omethoate. The working curve equation was △mP = 413.8 + 102.7lgc (pM) with detection limit of

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0.029 pM and correlation coefficient of 0.999.

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In order to investigate the selectivity of the sensor, the interference of coexisting ions and

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biomolecules was studied. in Fig. 5A. When the concentration of Na+, K+, Ca2+, Mg2+, Ba2+, Fe3+

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reached 1000 times of omethoate concentration, the concentration of Cu2+ and Ni2+ was 100 times,

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the concentration of L-proline, L-phenylalanine, RNA, DNA, vitamin B, lysozyme, albumin and

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lysine was 10 times, no obvious change in fluorescence intensity was observed. The effects of

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structurally similar pesticides were also investigated in Fig. 5B. The results shown that only

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omethoate could recover the fluorescence of the sensor. The other five pesticides had no

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significant effects on the fluorescence intensity, suggesting that the constructed sensor had good

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selectivity for omethoate pesticide.

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Figure 5

248 249

3.4 The determination of actual samples

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In order to evaluate the application of the sensor in actual samples, the Pearl River water and

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Chinese cabbage were selected as actual samples. Omethoate was not detected in water sample

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and cabbage sample, the standard addition method was used to determine the recovery rate. As

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illustrated in Table 1, when the added concentration of omethoate was 4.0 nM, the detected

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concentration by fluorescence analysis, fluorescence polarization method was consistent with

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HPLC, which suggested the excellent reliability of the fluorescence analysis and fluorescence

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polarization method. When the added concentration was 0.050 nM, omethoate could be detected

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only by fluorescence analysis and fluorescence polarization method, which suggested the

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sensitivity of these methods was superior to HPLC. When the added concentration was 0.10 pM,

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omethoate could be detected only by fluorescence polarization method, which suggested the

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sensitivity of fluorescence polarization method was further improved.

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Table 1 To visually detect the residual omethoate on the plants in situ, the GO...N-GQDs-aptamer 10

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probe was utilized for imaging of omethoate in Chinese cabbage leaves under fluorescence

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microscope. The control leaf, leaf with only omethoate, leaf with only GO...N-GQDs-aptamer

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probe shown the spontaneous yellow-green fluorescence of cabbage leaves in Figure 6A-C. When

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the leaves were treated with both omethoate and GO...N-GQDs-aptamer in Figure 6D-F, blue

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fluorescence was observed, and the intensity reduced with the decreasing concentration of

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omethoate. This meant the imaging could be used as visual semi-quantitative analysis of

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omethoate. When the concentration of omethoate soulution was as low as 0.1 µg/L which was

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much lower than 20 µg/kg from the food safety standard on omethoate in vegetables in China (GB

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2763-2012) , blue fluorescence could still be observed (Fig. 6F).

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Figure 6

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Compared with other omethoate detection methods reported in the last five years in Table 2,

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the detection limits of fluorescence analysis and fluorescence polarization analysis method in this

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paper were lowest. The method had high sensitivity, good selectivity, wide detection range.

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Furthermore, it could be used for visual semi quantitative determination of omethoate.

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Table 2

279 280

Acknowledgments: This work was supported by the National Natural Science Foundation of

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China (Nos. 21575043, 21275056, 21605052, 51478196); and the Platform Construction Project

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of Guangzhou Science Technology and Innovation Commission (No. 15180001).

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Tang, T. T.; Deng, J. J.; Zhang, M.; Shi, G. Y.; Zhou, T. S. Quantum dot-DNA aptamer

Song, A. Y. Determination of 13 Organic Toxicants in Human Blood by Liquid-Liquid

Tsoutsi, C.; Konstantinou, I.; Hela, D.; Albanis, T. Screening method for organophosphorus

Bakas, I.; Ben, O. N.; Moczko, E.; Istamboulie, G.; Piletsky, S.; Piletska, E.; Ait-Addi, E.;

Dou, X. W.; Chu, X. F.; Kong, W. J.; Luo, J. Y.; Yang, M. H. A gold-based nanobeacon probe

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chemiluminescence-molecular imprinting sensor for sequential determination of carbofuran and

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13. Catala-Icardo, M.; Lopez-Paz, J. L.; Choves-Baron, C.; Pena-Badena, A. Native vs

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photoinduced chemiluminescence in dimethoate determination. Anal. Chim. Acta. 2012, 710,

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14. Zhang, Y. Y.; Cao, T. C.; Huang, X. F.; Liu, M. C.; Shi, H. J.; Zhao, G. H. A Visible-Light

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Driven Photoelectrochemical Aptasensor for Endocrine Disrupting Chemicals Bisphenol A with

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High Sensitivity and Specificity. Electroanal. 2013, 25, 1787-1795.

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15. Sun, X.; Wang, X. Y. Acetylcholinesterase biosensor based on prussian blue-modified

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16. Pang, S.; Labuza, T. P.; He, L. L. Development of a single aptamer-based surface enhanced

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Surface Enhanced Raman Spectroscopy. Chinese J. Anal. Chem. 2010, 38, 1127-1132.

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Zhao, P. N.;

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J.;

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H. Multi-branch

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imaging of acetamiprid residues based on quantum dots functionalized with aptamer. Sensor.

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Actuat. B-Chem. 2016, 229, 100-109.

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22. Jia, L.; Ding, L.; Tian, J. W.; Bao, L.; Hu, Y. P.; Ju, H. X.; Yu, J. S. Aptamer loaded MoS2

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nanoplates as nanoprobes for detection of intracellular ATP and controllable photodynamic therapy.

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Nanoscale. 2015, 7, 15953-15961.

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23. Liu, Q. L.; Xu, S. H.; Niu, C. X.; Li, M. F.; He, D. C.; Lu, Z. L.; Ma, L.; Na, N.; Huang, F.;

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Jiang, H.; Ouyang, J. Distinguish cancer cells based on targeting turn-on fluorescence imaging by

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folate functionalized green emitting carbon dots. Biosens. Bioelectron. 2015, 64, 119-125.

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24. Wang, D.; Lin, B. X.; Cao, Y. J.; Guo, M. L.; Yu, Y. A Highly Selective and Sensitive

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Fluorescence Detection Method of Glyphosate Based on an Immune Reaction Strategy of Carbon

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Dot Labeled Antibody and Antigen Magnetic Beads. J. Agric. Food Chem. 2016, 64, 6042-6050.

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32. Qi L.; Yan Z.; Huo Y.; Hai X. M.; Zhang Z. Q. MnO2 nanosheet-assisted ligand-DNA

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interaction-based fluorescence polarization biosensor for the detection of Ag+ ions. Biosens.

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into Blue-Luminescent Graphene Quantum Dots. Adv. Mater. 2010, 22, 734-738.

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Biosens. Bioelectron. 2015, 68, 225-231.

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Figure captions

386

Scheme. The detection mechanism based on the N-GQDs-aptamer probe.

387

Figure 1. The synthesis conditions optimization of N-GQDs (A) and N-GQDs-aptamer probe (B).

388

A1: the doping amount (mA and mB respectively for urea and graphene oxide); A2: pH; A3:

389

reaction time; A4: reaction temperature; B1: the coupled aptamer amount (mA and mN are the

390

amount of aptamer and N-GQDs); B2: pH and reaction time.

391

Figure 2. Characterization of N-GQDs and N-GQDs-aptamer. A: excitation and emission spectra,

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curves 1-3 represented GQDs, N-GQDs and N-GQDs-aptamer, respectively. B: the fluorescence

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polarization spectra. C: TEM images of N-GQDs (C1) and N-GQDs-aptamer (C2). The

394

concentration of N-GQDs or N-GQDs-aptamer was 0.279 mg/ml.

395

Figure 3. Quenching of N-GQDs-aptamer by different materials (A1: carbon dots, A2: carbon

396

nanotube, A3: GO) and recovery by omethoate (A). Curve 1, 2, 3 represented respectively

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N-GQDs-aptamer,N-GQDs-aptamer + quencher,N-GQDs-aptamer + quencher + omethoate,the

398

concentrations of quencher and omethoate were 0.625 mg/ml and 10 nM respectively. The effects

399

of different conditions on the quenching of N-GQDs-aptamer (B). (B1: GO amount, B2:

400

quenching time, B3: The quenching effect of GO on N-GQDs and N-GQDs-aptamer), the

401

concentration of N-GQDs or N-GQDs-aptamer was 0.279 mg/ml.

402

Figire 4. Fluorescence spectra of 1: omethoate; 2:N-GQDs-aptamer; 3:GO; 4: 1+2; 5:1+3; 6:2+3;

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7:1+2+3 and UV-visual spectra of GO (A). TEM of GO (B), B1: GO; B2: GO+N-GQDs-aptamer;

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B3: GO+N-GQDs.

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Figure 5. Interference of coexisting ions, biomolecules (A) and structurally similar pesticides (B).

406

Figure 6. Imaging of Chinese cabbage leaves. A: blank; B: leaf with omethoate; C: leaf with

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GO...N-GQDs-aptamer; D-F: leaf with both GO...N-GQDs-aptamer and omethoate at

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concentration of 20, 5, 0.1 µg/L.

409 410 411 412

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Table 1 Determination of omethoate in river water and cabbage

Fluorescence analysis Samples

Pearl river water

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Fluorescence polarization

Added Detected

Recovery

RSD

Detected

(nM)

(%)

(%)

(nM)

0

ND





0.10 pM

ND



0.050

0.0475

4.0

Recovery

HPLC Recovery

RSD

Detected

(%)

(%)

(nM)

(%)

(%)

ND





ND







0.0912 pM

91.2

6.0

ND





95.0

7.1

0.0489

97.8

4.8

ND





3.76

94.0

4.1

3.83

95.7

3.6

3.58

89.5

6.3

0

ND





ND





ND





0.10 pM

ND





0.0958 pM

95.8

5.0

ND





0.050

0.0496

99.2

8.0

0.0492

98.4

4.0

ND





4.0

3.94

98.5

3.6

3.78

94.5

4.3

4.01

100.2

5.4

(nM)

RSD

cabbage

414

ND: not found

415 416

Table 2 Comparison of omethoate detection methods Detection methods

Detection range (nM)

Detection limit (nM)

-1

literature

-2

this paper *

cabbage, Pearl

1.0×10 -1.7×10

4.1×10

this paper **

River water

1.0×10-4-1.0×103

2.9×10-5

2.7×10-2 -2.7×104

2.3×103

[9]

carbohydrate



2.3×10

[10]

apple juice



2.4×105

[16]

fruits

3.0×102 -1.0×104

2.3×102

[5]

water



2.3×10-1

[13]

4.7×102 -4.2×104

1.2×102

[12]

fluorescence fluorescence polarization surface enhancement capillary electrophoresis light induction chemiluminescence

417

Samples

orange peel, running water

pears, carrots, eggplant

* fluorescence analysis method

** fluorescence polarization method

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