A Highly Selective and Sensitive Fluorescence Detection Method of

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A highly selective and sensitive fluorescence detection method of glyphosate based on an immune reaction strategy of carbon dots labeled antibody and antigen magnetic beads Duo Wang, Bixia Lin, Yujuan Cao, Manli Guo, and Ying Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01088 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 13, 2016

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Journal of Agricultural and Food Chemistry

Title: A highly selective and sensitive fluorescence detection method of glyphosate based on an immune reaction strategy of carbon dots labeled antibody and antigen magnetic beads

Authors and affiliations: Duo Wang, Bixia Lin, Yujuan Cao, Manli Guo, Ying Yu*

School of Chemistry and Environment, South China Normal University, Guangzhou, Key Laboratory of Analytical Chemistry for Biomedicine, Guangzhou, Guangdong, 510006, 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, China, 510006

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ACS Paragon Plus Environment


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A highly selective and sensitive fluorescence detection method of

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glyphosate based on an immune reaction strategy of carbon dots

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labeled antibody and antigen magnetic beads

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Duo Wang, Bixia Lin, Yujuan Cao, Manli Guo, Ying Yu*

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School of Chemistry and Environment, South China Normal University, Guangzhou,

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Key Laboratory of Analytical Chemistry for Biomedicine, Guangzhou, Guangdong,

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510006, China.

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Abstract

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A sensitive fluorescence detection method for glyphosate (GLY) was established

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based on immune reaction. Firstly, carbon dots labeled antibody (lgG-CDs) which

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were able to specifically identify glyphosate were prepared with the environmental

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friendly carbon dots (CDs) and glyphosate antibody (lgG). lgG-CDs could be used to

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in situ visualize the distribution of glyphosate in plant tissues. In order to eliminate

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the effects of excess lgG-CDs on the determination of GLY, antigen magnetic beads

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Fe3O4-GLY based on magnetic nanoparticles Fe3O4 and glyphosate were constructed

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and utilized to couple with the excess lgG-CDs. After magnetic separation to remove

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antigen magnetic beads, there was a liner relationship between the fluorescence

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intensity of lgG-CDs and the logarithmic concentration of glyphosate in the range of

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0.01~80 µg/mL with a detection limit of 8 ng/mL. The method was used for the

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detection of glyphosate in Pearl River water, tea and soil samples with satisfactory

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recovery ratio between 87.4%~103.7%.

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Keywords: glyphosate; carbon dots; magnetic beads; fluorescence immunoassay;

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quantitative determination

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1. Introduction

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Glyphosate is a widely used herbicide, which has threat to human health through

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the residue in food and has harmful effects on the ecology as it is released into the

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environment1. Therefore, more and more attention is being focused on the detection of

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glyphosate in crops and environment. It is significant to establish a sensitive, selective,

46

simple and convenient detection method for glyphosate.

47

Chromatography and chromatography combined with mass spectrometry were

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the common methods for the detection of glyphosate2-7. Due to the high polarity and

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low molecular mass of glyphosate, complex derivatization process was often requisite

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to acquire enough column retention for chromatography and chromatography coupled

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with mass spectrometry. For example, glyphosate was derivatized by trifluo-acetic

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anhydride8,

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detecting by chromatography. To avoid such complex derivatization process, ion

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chromatography was also used to determinate glyphosate10,11. However, the selectivity

55

of ion chromatography was limited.

trifluoroethanol8,

and

4-chloro-3,5-dinitrobenzotrifluoride9

before

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In addition, capillary electrophoresis12-14, electrochemical method15,16, surface

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resonance enhanced spectrometry17, enzyme linked immunoassay18-22 and fluorescent

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spectrometry were also reported to detect glyphosate in current literatures. The

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detection limit of ELISA was higher than HPLC19 and LC-MS20,21. For example, a

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linker-assisted enzyme-linked immunosorbent assay (L’ELISA) was established for

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glyphosate with detection limit of 0.1 µg/L. The target (glyphosate) was required to

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derivatize in advance. The results of L’ELISA were false positive rate of 18% 4


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between 0.1 and 1.0 µg/L and a false positive rate of only 1% above 1.0 µg/L 21.

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Based on a competitive enzyme-linked immunoassay using antibody specific to

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glyphosate, Selvi et al.22 utilized phosphatase as marker enzyme and established the

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method for the measurement of glyphosate derived from coffee, rice and other food

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samples. The assay was specific to glyphosate with a detection limit of 2 ppb. But the

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average recoveries for different food samples were varied from 90.00% to 134.00%.

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These might be that the enzyme was vulnerable to many factors and became inactive,

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which made the determination results of low accuracy and poor reproducibility. Guo23

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designed a fluorescence assay for glyphosate. The fluorescence of CdTe was

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quenched due to the fluorescence resonance energy transfer between negatively

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charged CdTe and positively charged gold nanoparticles. Under the appropriate

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acidity, the introduction of negatively charged glyphosate would replace CdTe, thus

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the fluorescence of CdTe would recover. This fluorescence assay was based on the

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nonspecific electrostatic attract and thus the selectivity was limited.

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Because of the high selectivity, sensitivity and easy in-situ detection, the

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fluorescence immunoassay received great attention. Carbon dots, as a new type of

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fluorescent material, were reported to be used for constructing fluorescent probes to

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detect metal ions24, label and image tumor cells25,26. Ouyang group27 designed a

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fluorescent probe for targeting imaging of cancer cells via hydrogen-bond interaction

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between folic acid and CDs that could accurately distinguish folate receptor of

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positive cancer cells in different cell mixtures. Zhu28 used Fe3+/Fe2+ to turn off / turn

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on the fluorescence of carbon dots for imaging study of mouse embryonic osteoblasts. 5



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In the immunoassay, the matrix in sample solutions was often more complex. In order

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to eliminate the interference from matrix and improve the signal to noise ratio of the

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detection, magnetic nanoparticles such as Fe3O4 after modified by antibody, aptamer

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and the like could be utilized to separate and enrich the analytes under the magnetic

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field via combining with the analytes. Su group29 established a sensitive method for

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detection of avian influenza virus antigen based on the magnetic nanoparticles

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modified with avian influenza virus antibody. Nie group30 used aptamer-conjugated

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magnetic beads and a magnet-quartz crystal microbalance to establish a method for

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selective collection and detection of human acute leukemia cells. Ju group31 used

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alpha fetoprotein antibody-conjugated magnetic beads to separate the alpha fetal

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protein and the cancer embryo protein from the mixture. Then the above two types of

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proteins were quantified with chemiluminescence immunoassay.

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In this paper, lgG-CDs were constructed by coupling the environmental friendly

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carbon dots and glyphosate antibody. The as-prepared lgG-CDs could specifically

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identify glyphosate and in situ visualize the distribution of glyphosate in the plant

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tissues. In addition, antigen magnetic beads Fe3O4-GLY were assembled and added

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into the solution to combine with excess lgG-CDs through competitive immune

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reaction, subsequently the antigen magnetic beads were separated from the solution

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by the magnetic field and thus the effects of excess lgG-CDs on the determination of

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GLY were eliminated. Meanwhile, a linear relationship between the logarithmic

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concentration of glyphosate and fluorescence intensity of lgG-CDs in solution was

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identified, according to which, the quantitative detection of glyphosate in real samples 6


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was realized. The determination principle for GLY was shown in Scheme 1. The

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determination results suggested the method was sensitive, selective, simple and

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convenient compared with the prior methods reported in the literatures. Scheme 1

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2. Materials and methods

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2.1. Materials and instruments

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Citric acid, NaCl, Na2HPO4·12H2O and NaH2PO4·2H2O were purchased from

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Tianjin Zhiyuan Chemical Reagent Company. Ethidene diamine, glyphosate

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(N-(Phosphonomethyl)glycine), carbodiimide hydrochloride (EDC·HCl, 98.5%),

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N-hydroxysuccinimide (NHS, 98%), amino-modified Fe3O4 (Fe3O4,-NH2) with

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particle size of 100 nm~200 nm were purchased from Aladdin Chemical Company.

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The soil was purchased from JIFFY Company, which particle size is 0-8 mm, the pH

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is 5.2-6.0, electrical conductivity is 1. All chemicals used were of analytical grade or

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of the highest purity available. Double-distilled water was used throughout the

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experiments.

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Fluorescence spectra were obtained using an F-4500 fluorometer (Hitachi, Japan)

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with a voltage of 400 V, a slit width of 5 nm and a scanning speed of 300 nm /min.

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UV-vis absorption spectra were measured with a 1700 UV-vis spectrophotometer

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(Techcomp, China). Fluorescence lifetime experiments were performed by an

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FLS-920 combined fluorescence lifetime and steady-state spectrometer (Edinburgh,

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UK). Hydrated sizes of particles were measured using Nano ZS nanometer particle

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size analyzer (Malvern, UK). Fourier transformed infrared (FTIR) spectra were

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acquired over the range of 500-4000 cm-1 with IR Prestige-21 spectrophotometer

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(Shimadzu, Japan) and KBr crystals as the matrix for sample preparation.

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Thermogravimetric

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thermogravimetric analyzer. The imaging of cabbage seedling roots was obtained by

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LSM 510 Meta Duo Scan laser scanning confocal microscope (Carl Zeiss, Germany).

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2.2. Preparation of glyphosate antigen and antibody

(TG)

data

were collected with

SPA409PC

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As indicated in a reported study32, complete antigen (GLY-BSA) was

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synthesized through the coupling reaction of glyphosate and bovine serum albumin.

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Subsequently, the emulsion including glyphosate antigen and the same volume of

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Freund's adjuvant was injected into New Zealand white rabbits by subcutaneous

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injection. After 21 days, enhancement immunity was performed every 14 days and

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repeated 2~3 times. As the antibody titer was qualified, the glyphosate antibody was

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obtained by centrifugation and salting-out of the carotid blood samples. The lgG

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concentration was 12.7 mg/mL and the bidirectional diffusion titer was 1:32.

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2.3. Preparation of carbon dots

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Carbon dots were prepared according to a reported method28 with a little

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modification. In detail, 3.0 g citric acid was dissolved by 30 mL of 1.0 M

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ethylenediamine. Then the solution was transferred into a 100 mL Teflon-lined

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stainless steel autoclave and heated at 200 °C. After 3 h, the reactor was cooled to

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room temperature. The reactant was loaded into 3.5 KDa dialysis bag and then 8


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dialyzed by double-distilled water once per 30 min until the conductivity of dialyzate

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closed to that of double-distilled water. After cryodesiccation, 2.37 g CDs were

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obtained and dissolved by 0.01 M PBS (pH=7.4). The final concentration of CDs was

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80 mg/mL.

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2.4. Preparation of lgG-CDs

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lgG-CDs were prepared according to a reported method33 with a little

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modification. 380 mM EDC and 380 mM NHS were prepared with 0.1 M PBS ( pH

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=7.4 ) as solvent. 4.0 mL of 380 mM EDC and 4.0 mL of 380 mM NHS were added

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in 25 mL three-necked flask and stirred, then 1.0 mL of 80 mg/mL prepared CDs

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were added. The mixed solution was stirred under nitrogen at 37 °C for 15 min.

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Subsequently, 5 µL of 12.7 mg/mL glyphosate antibody was added into the above

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mixture and stirred for 2 h. The resulting solution was refrigerated at 4 °C in the dark

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to stop the reaction. Eventually, the solution was loaded into 3.5 KDa dialysis bag and

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dialyzed by double-distilled water once per 30 min until the conductivity of dialyzate

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closed to that of double-distilled water. The lgG-CDs obtained was stored at 4 °C in a

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refrigerator and the concentration counted according to glyphosate antibody was

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6×10-3 mg/mL.

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2.5. Imaging of GLY residues in cabbage seedling root

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Cabbage seedling roots cultured for one month were cut into pieces. The tissue

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sections were incubated with 10 mL of double-distilled water or 0.1 µg /mL GLY for

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1 h, subsequently washed by double-distilled water and incubated with 10 mL of 3 9



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µg/mL CDs solution at 25 ℃in the darkness for 1 h. Finally, the tissue sections were

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washed three times with double-distilled water. Some other sections of cabbage roots

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were performed following the same procedure above but incubated with 10 mL of

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CDs-lgG (the concentration according to CDs was 3 µg/mL) instead of CDs. All the

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treated tissue sections were observed between 410 to 500 nm wavelength range under

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laser confocal microscope.

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2.6. Preparation of antigen magnetic beads Fe3O4-GLY

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5.0 mL of 160 mM EDC, 5.0 mL of 160 mM NHS and 5 mL of 8 mg/mL GLY

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were added in 100 mL three-necked flask and stirred under dry nitrogen at 37 °C for

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15 min. Then, 20 mL of 50 mg/mL Fe3O4-NH2 was added into the above mixture and

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stirred for 3 h. After magnetic separation, the solid was washed by double-distilled

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water for 3 times and dried under vacuum environment at 50 °C. Finally, 0.93 g

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Fe3O4-GLY were obtained and suspended by 0.01 M PBS (pH=7.4). The final

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concentration of Fe3O4-GLY was 10 mg/mL.

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2.7. Determination of GLY

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Real samples were processed as follows: 100 mL of water samples from the

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Pearl River was added with GLY solution and centrifuged with 4000 rmp for 5 min.

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And then the supernatant was filtered with 0.22 µm filter membrane and diluted to

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100 mL accurately with double-distilled water. Tea or soil were added with GLY

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solution, and then dried naturally, crushed and sieved with 60 mesh sieve. Thereafter，

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1.0 g crushed tea powder or sieved soil was added by 5 mL of double-distilled water 10


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and extracted under room temperature. After 2 h, the mixture was filtered and washed

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with distilled water for 3 times. Finally, the filtrate was filtered again by 0.22 µm

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membrane and diluted accurately to 100 mL.

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100 µL of 6×10-3 mg/mL lgG-CDs were added in 5 mL colorimetric tubes，0.5

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mL of different concentrations of GLY standard solution or real samples, 2 mL of 10

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mg/mL Fe3O4-GLY including 3×10-3 mmol glyphosate were added in turn and diluted

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to 5 mL with 0.01 M PBS ( pH =7.4 ). After incubated at 37 °C for 40 min, the

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reactants were separated by magnet and the supernatant was detected by fluorescence

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spectrometer under slit of 5 nm, voltage of 400 V and λex/λem of 344/444 nm. Finally,

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according to the fluorescence intensity of supernatant, the working curve was drawn

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and the concentrations of GLY in real samples were calculated by the working curve.

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3. Results and discussion

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3.1. Characterization of lgG-CDs and imaging of GLY

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The lgG-CDs prepared according to experiment method 2.4 were characterized

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by UV-vis spectrometry, fluorescence spectrometry, fluorescence lifetime analysis,

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dynamic light scattering analysis and so on.

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UV-vis spectra were shown in Fig. 1a. CDs had a strong absorption peak at 345

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nm. When there was no coupling agents EDC and NHS, the mixture of lgG and CDs

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was dialyzed by 16 KD dialysis bag and CDs with low molecular weight would go

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through the dialysis bag into the dialysate, so only the characteristic absorption peak

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of lgG at 280 nm was observed. But as coupling agents EDC and NHS were

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introduced, CDs combined with lgG to form lgG-CDs with large molecule weight that

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couldn’t go through the dialysis bag, so the absorption peak of lgG-CDs appeared at

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351 nm which was 6 nm red-shift by compared with that of CDs. The difference of

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absorption peaks proved that CDs were coupled with lgG .

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The fluorescence properties of lgG-CDs were characterized by fluorescence

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spectrometry. As could be seen from Fig. 1b, both CDs and lgG-CDs exhibited a

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broad excitation peak between 320 to 380 nm. Compared with the emission

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wavelength of CDs at 444 nm, the fluorescence peak of lgG-CDs was red-shifted

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about 13 nm to 457 nm. This also indicated that CDs were coupled with lgG.

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The fluorescence decay curves could be applied to characterize the behavior of

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molecule or particle at excited state. In Fig. 1c, the fluorescence decay curves of CDs,

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the mixture of CDs and lgG, lgG-CDs conjugation were shown respectively and the

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fluorescence average lifetimes were calculated in embedded table. From the table, it

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could be seen that fluorescence lifetimes (τ) of CDs and the mixture of CDs and lgG

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were 25.26 and 25.03 ns which had little difference. This shown that the luminophor

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in both of the two samples was CDs, which confirmed the simple mixing of CDs and

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lgG did not form new substance. However, the fluorescence lifetime of lgG-CDs was

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21.77 ns, which was obviously shorter than that of CDs. The difference in lifetime

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suggested the behavior of CDs at excited state was varied from that of lgG-CDs and

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the forming of lgG-CDs was further comfirmed.

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The photobleaching property of lgG-CDs was determined. Both the fluorescence

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of CDs and lgG-CDs were recorded at λex/λem = 344/444 nm while fluorescein (FITC) 12


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was recorded at λex/λem = 490/525 nm. As displayed in Fig. 1d, the fluorescence

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intensity of lgG-CDs was only decreased by 2.68% after exposure for 3600 s, while

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FITC was greatly decreased by 34.4% after exposure for 500 s. It suggested that

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lgG-CDs had stronger resistance to photobleaching. Besides, relative fluorescence

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quantum yield of lgG-CDs was 56.7% which closed to 57.1 % of CDs synthesized in

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this paper. Thus the synthesized lgG-CDs could still maintain the excellent

240

fluorescence property as CDs.

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The particle sizes of CDs, lgG, the mixture of CDs and lgG, and lgG-CDs could

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be analyzed by Malvin particle size analyzer. As shown in Fig. 1e, the size

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distribution of CDs was in the range of 5~8 nm and lgG was in the range of 7~10 nm.

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When CDs mixed with lgG in the absence of coupling agent, the particle size was

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mainly distributed in the range of 11~15 nm and the range became wider. This might

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be due to the adsorption and aggregation appeared in the mixture. However, the

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particle size of lgG-CDs was distributed around 200 nm that was much greater than

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any of the above three samples. It was shown that CDs and lgG were coupled

249

successfully under the action of coupling agent.

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Fig. 1

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According to experiment method 2.5, lgG-CDs obtained were utilized to directly

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image for GLY in cabbage seedling root sections. It could be seen from Fig. 2, when

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the root tissue sections were incubated respectively with CDs (Fig. 2a), GLY and CDs

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(Fig. 2b), lgG-CDs (Fig. 2c), only weak fluorescence could be observed. This was

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attributed to the nonspecific adsorption of CDs or lgG-CDs by the tissues. However, 13



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as the root tissue sections were incubated with both GLY and CDs-lgG (Fig. 2d), the

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fluorescence intensity was greatly enhanced and there were various fluorescence

258

intensities in different regions of the tissues. This proved that CDs-lgG could

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specifically identify and image for GLY in the plant tissues, which could be applied

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for in-situ visualization detection of GLY pesticides.

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Fig. 2 3.2. Synthesis and characterization of Fe3O4-GLY

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In order to eliminate the interference of excess lgG-CDs on the determination of

264

GLY, antigen magnetic beads Fe3O4-GLY which could combine with excess lgG-CDs

265

via antigen-antibody recognition were designed and synthesized. After magnetic

266

separation, Fe3O4-GLY and excess lgG-CDs were removed from the solution, and

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therefore the fluorescence interference derived from excess lgG-CDs was eliminated.

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Generally speaking, the more GLY was bound to magnetic beads Fe3O4, the

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more advantageous would be for Fe3O4-GLY to combine with the free lgG-CDs. Thus

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with the increasing of the concentration of GLY added, the amount of GLY bound to

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the magnetic beads was increased gradually and finally reached saturation with

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maximum binding ratio. However if the concentration of GLY was increased

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unceasingly after saturation, excess GLY would be adsorbed on the surface of

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magnetic beads and desorbed in the sample solutions to combine with free lgG-CDs.

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This would cause the increase of fluorescence intensity in supernatant, which made

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the test value higher than the true value of analyte. Thus the mass ratio of GLY and

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magnetic beads was optimized. When Fe3O4-GLY with different mass ratios of GLY 14


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and magnetic beads were added into the lgG-CDs solution, the fluorescence intensity

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of the supernatant after magnetic separation was determined and the results were

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shown in Fig. 3a. As the mass ratio of GLY and magnetic beads was 1:25,

281

fluorescence intensity of the supernatant was lowest. So the optimal mass ratio of

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GLY and magnetic beads was 1:25.

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Fe3O4-GLY synthetized under the optimal mass ratio of Fe3O4 and GLY were

284

confirmed by infrared spectrometry in Fig. 3b. As could be seen from the FTIR

285

spectrum of Fe3O4,-NH2, stretching vibration and bending vibration peaks of N-H

286

bond appeared at 3451 cm-1 and 1640 cm-1. Another strong absorption band was at

287

593 cm−1, which was attributed to the bending vibration peak of the Fe-O bond. In the

288

FTIR spectrum of GLY, the peaks at 1720 cm-1 and 3014 cm-1 were the characteristic

289

absorption of carboxyl group and its dimers, which shown that GLY contained

290

abundant -COOH group. In the FTIR spectrum of Fe3O4-GLY, the absorption peaks

291

of carboxyl group in GLY and amino group in Fe3O4,-NH2 disappeared because they

292

were coupled to form amide bond which shown absorption peak at 1651 cm−1. These

293

proved that Fe3O4-GLY were sucessfully synthetized.

294

The amount of GLY bound to magnetic beads was verified by thermogravimetry

295

analysis. Fig. 3c indicated both the weights of Fe3O4 and Fe3O4-GLY appeared stable

296

after 600 °C. Fe3O4 had weight loss of 6.8 % which was attributed to the

297

decomposition of amino silane on the surface of Fe3O4. However Fe3O4-GLY had

298

weight loss of 9.4 % which was caused by the decomposition of both amino silance

299

and GLY. Therefore, it could be calculated that the percentage of GLY in Fe3O4-GLY 15



300

was about 2.6%.

301

The magnetic separation performance of antigen magnetic beads towards

302

lgG-CDs was confirmed intuitively by the magnetic separation experiments. As could

303

be seen from Fig. 3d, in the absence of Fe3O4-GLY and magnetic field, lgG-CDs

304

presented strong fluorescence under UV light. After adding Fe3O4-GLY, the solution

305

still maintained stable and uniform with strong fluorescence. However, when the

306

magnetic field was also introduced, Fe3O4-GLY combined with lgG-CDs was

307

gathered to the magnet aside, thus fluorescence was observed only in the magnet aside.

308

This revealed that Fe3O4-GLY could be used for magnetic separation of lgG-CDs.

309

310

Fig. 3 3.3. Optimization of determination condition for GLY

311

It was found that the fluorescence intensity from excess lgG-CDs in the solution

312

disturbed the detection of GLY. So it was necessary to remove the excess lgG-CDs.

313

Because of the specific recognition of lgG-CDs to GLY, Fe3O4-GLY was introduced

314

to competie with the target analyte for antibody recognition, and separate the excess

315

lgG-CDs. The determination principle was shown in Scheme 1. Briefly, the lgG-CDs

316

solution was added successively with GLY and excess Fe3O4-GLY. Partial lgG-CDs

317

were combined with GLY. The other lgG-CDs would combined with Fe3O4-GLY and

318

be removed by magnetic separation. Then the fluorescence intensity of supernatant (F)

319

containing the conjugation of lgG-CDs and GLY was determined. The quantitative

320

determination method of GLY was established based on the relationship between F

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and the concentration of GLY.

322

In order to ensure the activity of antibody, the determination was carried out in

323

pH 7.4 and 37 °C. The other conditions such as the mass ratio of Fe3O4-GLY and

324

lgG-CDs and the reaction time were optimized. In Fig. 4a, when the concentration of

325

GLY was in the range of 0.01~80 µg/mL, the optimal mass ratio of lgG-CDs and

326

Fe3O4-GLY was (3×10-5):1. Besides, with the extending of reaction time, the

327

fluorescence intensity of supernatant increased and tended to be stable after 40 min,

328

so 40 min was selected as the optimal reaction time (Fig 4b).

329

Under the optimal conditions, the working curve was obtained according to

330

experiment method 2.7. As showed in Fig. 4c, when the concentrations of GLY were

331

in the range of 0.01~80 µg /mL, the fluorescence intensity of supernatant was linearly

332

related to the logarithm concentration of GLY. The working curve was F=2099+1022

333

lg CGLY (CGLY was the concentration of GLY, µg/mL) and the detection limit was 8

334

ng/mL.

335

336

Fig. 4 3.4. Selective experiments of the method

337

To verify the specific recognition of the proposed method, some pesticides with

338

the similar structure such as pmida, glyphosine, omethoate and frequently-used

339

phosmet were employed for control studies. As illustrated in Fig. 5a, when the

340

concentration of glyphosate was 5 µM and other pesticides were 50 µM, the

341

fluorescence intensities of pmida and glyphosine were slightly higher than those of

17



342

phosmet and omethoate, which was because the structures of the former two

343

pesticides were more similar with GLY. However, by compared with GLY, all the

344

fluorescence intensities of these four pesticides were very low. The above results

345

suggested that this competitive immunoassay based on lgG-CDs enabled highly

346

selective recognition of GLY.

347

The interference of common coexistent ions on the fluorescence intensity of the

348

system was examined. Fig. 5b showed the interference of various ions including Na+,

349

K+, NH4+, NO3-, P043- and F- with a concentration of 40 mM, Mg2+, Zn2+, Ca2+ and

350

Fe3+with a concentration of 2 mM. The change of fluorescence intensity was not

351

observed in the presence of these metal ions except for Fe3+ when 5 µM of GLY was

352

detected. The fluorescence intensity was greatly reduced in the presence of Fe3+,

353

which was due to the quenching effect of Fe3+ on the CDs of lgG-CDs 28. When Fe3+

354

was masked by F- ion, the fluorescence intensity of the solution was close to the

355

control group. The results suggested that established method had strong

356

anti-interference ability and could be used for detection of GLY in complex samples.

357

358

Fig. 5 3.5. Determination of GLY in real samples

359

To further investigate the applications of the proposed method, the amounts of

360

GLY in different samples from the Pearl River water, tea and soil were determined by

361

this method and ion chromatography 11. As shown in Table 1, the results measured by

362

this method were consistent with those by IC, which indicated this method had good

363

accuracy. The recovery ratio of this method was in the range from 87.4% to 103.7%, 18


Page 18 of 34

Page 19 of 34


364

and higher than that of IC. The RSD value was no more than 4.67, which indicated

365

this method had good repeatability. Table 1

366

367

Some detection methods of glyphosate reported in recent years were listed in

368

Table 2. The sensitivity reported by Guo23 was highest. However, according to the

369

measurement principle, any substances which could electrostatically adsorb gold

370

nanoparticles could be determined. Compared with these methods listed, the

371

fluoroimmunoassay method established in this paper was simple, selective and

372

sensitive with wide measurement range. Furthermore, precious instruments were not

373

needed. This method was a useful supplement for the traditional detection methods

374

such as chromatography and chromatography combined with mass spectrometry. Table 2

375

376

377

Acknowledgements

378

This work was supported by the National Natural Science Foundation of China (Nos.

379

21275056, 21575043, 51478196), the Platform Construction Project of Guangzhou

380

Science Technology and Innovation Commission (No. 15180001) and the cultivation

381

foundation of South China Normal University for young teachers (No.14KJ08).

382 383 384 385

19



386 387 388

Page 20 of 34

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cleanup by cation exchange resin. J. Sep. Sci. 2010, 33, 1139-1146.

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(9) Qian, K.; Tang, T.; Shi, T.; Li, P.; Li, J.; Cao, Y. Solid-phase extraction and

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residue determination of glyphosate in apple by ion-pairing reverse-phase liquid

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chromatography with pre-column derivatization. J. Sep. Sci. 2009, 32, 2394-2400.

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Garrido, T.; Farréet, M. Determination of glyphosate in groundwater samples using an

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ultrasensitive immunoassay and confirmation by on-line solid-phase extraction

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energy transfer between oppositely charged CdTe quantum dots and gold

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probes for rapid, sensitive, and label-free detection of Hg2+ and biothiols in complex

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sensors, and bioimaging. Angew. Chem.-Int. Edit. 2013, 52, 3953-3957.

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Selective collection and detection of leukemia cells on a magnet-quartz crystal

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496

(32) Clegg., B. S.; Stephenson., G. R.; Hall., J. C. Development of an enzyme－

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linked immunosorbent assay for the detection of GLY phosate. J. Agric. Food Chem.

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1999, 47, 5031-5037.

499

(33) Zhu, L.; Cui, X.; Wu, J.; Wang, Z.; Wang, P.; Hou, Y.; Yang, M. Fluorescence

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502

(34) Schrübbers, L. C.; Masís-Mora, M.; Rojas, E. C.; Christensen, J. H.;

503

Cedergreen, N. Analysis of glyphosate and aminomethylphosphonic acid in leaves

504

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506

(35) Rojano-Delgado, A. M.; Ruiz-Jiménez, J.; Castro, M. D. L. D.; Prado, R. D.

507

Determination of glyphosate and its metabolites in plant material by reversed-polarity

508

CE with indirect absorptiometric detection. Electrophoresis, 2010, 31, 1423–1430.

509

(36)Ding, X.; Yang, K. L. Development of an oligopeptide functionalized surface

510

plasmon resonance biosensor for online detection of glyphosate. Anal. Chem. 2013,

511

85, 5727-5733.

512

(37) De Almeida, L. K. S.; Chigome, S.; Torto, N.; Frostc, C. L.; Pletschke B. I. A

513

novel colorimetric sensor strip for the detection of glyphosate in water. Sens.

514

Actuators, B 2015, 206, 357–363.

515 516

25



517

Figure captions

518 519

Scheme 1. The detection principle of GLY.

520 521

Fig. 1. Characterization of lgG-CDs. a: UV-vis spectra of CDs (1), lgG-CDs (2) and

522

lgG (3); b: fluorescence spectra of CDs (1) and lgG-CDs (2); c: fluorescence lifetime

523

decay analysis of CDs (1), lgG-CDs (2), mixture of lgG and CDs (3); d:

524

photobleaching properties of CDs (1), lgG-CDs (2) and FITC (3), CDs and lgG-CDs

525

were recorded at λex/λem = 344/444 nm, FITC at λex/λem = 490/525 nm;. e: particle

526

size analysis of CDs (1), lgG (2), the mixture of CDs and lgG (3) and lgG-CDs (4).

527 528

Fig. 2. Fluorescence imaging of plant tissues treated with CDs (a); GLY and CDs (b);

529

lgG-CDs (c); GLY and lgG-CDs (d). All the treated tissue sections were observed

530

between 410 to 500 nm wavelength ranges. All the scale bars were 50 µm.

531 532

Fig. 3. Synthesis and characterization of Fe3O4-GLY. a: optimization of the mass ratio

533

of GLY and Fe3O4; b: infrared spectra of Fe3O4 (1), GLY (2) and Fe3O4-GLY (3); c:

534

TG analysis of Fe3O4 (1) and Fe3O4-GLY (2); d: magnetic separation experiment,

535

lgG-CDs in the absence of Fe3O4-GLY and magnetic field (1), lgG-CDs in the

536

presence of Fe3O4-GLY (2), lgG-CDs in the presence of both Fe3O4-GLY and

537

magnetic field (3).

538 26


Page 26 of 34

Page 27 of 34


539

Fig. 4. Optimization of determination condition. a: mass ratio, the concentration of

540

Fe3O4-GLY was 4 mg/mL ,and the GLY was (1) 80 µg/mL, (2) 1 µg/mL, (3) 0.01

541

µg/mL; b: reaction time of Fe3O4-GLY and lgG-CDs (the concentration of GLY was 1

542

µg/mL); c: the working curve of GLY by the presented detection method.

543 544

Fig. 5. The interference of pesticides (a) and coexistent metal ions (b), GLY with a

545

concentration of 5 µM, other pesticides with a concentration of 50 µM, Na+, K+,

546

NH4+, NO3-, P043-, F- with a concentration of 40 mM, Mg2+, Zn2+, Ca2+, Fe3+ with a

547

concentration of 2 mM. Fe3+* meant Fe3+ was masked with F- ion.

548

27



Table 1

549 550

Page 28 of 34

Detection of glyphosate in samples This method

Ion chromatography

Added samples

Found

Recovery

RSD (%)

Found

Recovery

RSD (%)

(µg/mL)

(%)

n=3

(µg/mL)

(%)

n=3

(µg/mL) nd

0

nd

Pearl

0.50

0.471

94.0

3.84

0.418

83.6

2.74

River

1.00

0.981

98.1

3.66

0.911

91.1

2.44

water

2.00

1.966

98.3

1.22

1.928

96.4

1.97

0

0.742

4.67

0.871

0.50

1.179

87.4

3.44

1.215

75.2

4.98

1.00

1.633

89.1

3.10

1.730

85.9

1.61

2.00

2.594

92.6

1.21

2.653

89.1

3.30

0

0.533

3.37

0.630

0.50

0.981

90.2

3.18

1.066

87.1

5.79

1.00

1.473

94.0

2.68

1.531

90.1

3.24

2.00

2.607

103.7

1.73

2.733

105.1

1.30

tea

soil

28


6.06

7.63

Page 29 of 34


Table 2 Comparison of determination methods reported in recent years

Method

Detection range

LOD

Samples

References

coffee leaves, HPLC/MS

56~520 µg/kg

12 µg/kg

rice, black beans,

34

river water Capillary electrophoresis Surface plasmon resonance

Colorimetry

L’ELISA

5~500 µg/mL

0.1 µg/mL

water

35

0.098 µg /mL

____

36

0.1~200 µg/mL

0.1 µg/mL

water

37

0.0004-0.01

0.0001

surface water

µg/mL

µg/mL

groundwater

0.02~2.0 µg/kg

0.0098 µg/kg

apples

0.01~80 µg/mL

0.008 µg/mL

0.084-10.82 µg/mL

21

Fluorescent spectrometry -CdTe/Au NPs

23

probe This method

river water, tea,

29


soil

____


Scheme 1. The detection principle of GLY.

30


Page 30 of 34

Page 31 of 34


Fig. 1. Characterization of lgG-CDs.

Fig. 2. Fluorescence imaging of plant tissues. 31



Fig. 3. Synthesis and characterization of Fe3O4-GLY.

32


Page 32 of 34

Page 33 of 34


Fig. 4. Optimization of determination condition.

Fig. 5. The interference of pesticides (a) and coexistent metal ions (b). 33



TOC Graph

34


Page 34 of 34

A Highly Selective and Sensitive Fluorescence Detection Method of

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