Sensitive and Facile Electrochemiluminescent Immunoassay for

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New Analytical Methods

A sensitive and facile electrochemiluminescent immunoassay for detecting genetically modified rapeseed based on novel carbon nanoparticles Hongfei Gao, Luke Wen, Yuhua Wu, Xiaohong Yan, Jun Li, Xiaofei Li, Zhifeng Fu, and Gang Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01080 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Journal of Agricultural and Food Chemistry

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Article

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A

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immunoassay for detecting genetically modified rapeseed

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based on novel carbon nanoparticles

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Hongfei Gao1,2, Luke Wen1, Yuhua Wu1, Xiaohong Yan1, Jun Li1, Xiaofei Li1, Zhifeng

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Fu3* and Gang Wu1*

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1

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Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China,

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sensitive

and

facile

electrochemiluminescent

Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops

National Key Laboratory of Crop Genetic Improvement and National Center of Plant

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Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China,

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3

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of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing

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400716, China.

Key Laboratory of Luminescence and Real-Time Analytical Chemistry of the Ministry

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___________________________________

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Correspondence and requests for materials should be addressed to

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G. Wu ([email protected]) or Z.F. Fu.( [email protected]).

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ACS Paragon Plus Environment


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ABSTRACT

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A highly sensitive electrochemiluminescent (ECL) immunoassay targeting PAT/bar

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protein was facilely developed for genetically modified (GM) rapeseed detection

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using carbon nanoparticles (CNPs) originally prepared from printer toner. In this work,

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CNPs linked with antibody for PAT/bar protein were used to modify a working

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electrode. After an immunoreaction between the PAT/bar protein and its antibody, the

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immunocomplex formed on the electrode receptor region resulted in an inhibition of

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electron transfer between the electrode surface and the ECL substance, thus led to a

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decrease of ECL response. Under the optimal conditions, the ECL responses linearly

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decreased as the increase of the PAT/bar protein concentration and the GM rapeseed

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RF3 content in the ranges of 0.10 - 10 ng/mL and 0.050% - 1.0%, with the limits of

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detection of 0.050 ng/mL and 0.020% (S/N=3). These results open a facile, sensitive

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and rapid approach for the safety control of agricultural GM rape.

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Keywords: immunoassay; electrochemiluminescence; genetically modified rapeseed;

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PAT/bar protein; carbon nanoparticles

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Introduction

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Genetically modified organisms (GMOs) obtain new valuable features by

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introducing exogenous genes via genetic engineering technology. Because of great

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application prospects in agriculture and food industry, genetically modified (GM)

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crops have been widely cultivated since the first GM variety was approved in 1996.1

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The global hectarage of GM crops has increased more than 100 times from 1.7

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million hectares in 1996 to 185.1 million hectares in 2015.2 The adoption of GM

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crops generates huge economic and social benefits, however, it also triggers the

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debate on the potential risk on the eco-environment when GM crops are released into

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the field. In particular, as a cross-pollinated plant, GM rape possesses the high natural

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crossing rate of 30% and tends to cross with other related wild species, resulting in

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transgene escape.3,4 In order to manage the ecological risk related to GM crops, new

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legislations and regulations are adopted, monitoring the production and application of

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GMOs in many countries. As the labeling policy of GM products was implemented,

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many countries stipulated their own labeling threshold, such as 0.9% in EU, 5% in

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Japan and 3% in Korea. The implementation of labeling regulations demanded the

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development of simple, rapid, accurate and low-cost analytical methods for GM crops

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screening, identification and quantification.

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The most widely-used GMO detection method is the DNA-based polymerase chain

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reaction (PCR) technique. Real-time quantitative PCR is currently recognized as gold

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standard method for GMO detection and quantification.

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than 100 real-time quantitative PCR methods are validated through ring trials and

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available in the database of “GMOMETHODS”.7 The PCR method, however,

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involves a complicated procedure including sample pretreatments, nucleic acid

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extraction, PCR preparation and amplification, electrophoresis of PCR products or 3


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In European Union more


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analysis of the fluorescent signal.8-10 This complicated procedure takes some hours to

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complete the detection of one sample.

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There is an increased attention for alternative, cheap and simple GMO detection

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technologies that successfully detect GM-derived material. One alternative strategy is

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to use certain affinity ligands comprising aptamers11,12 or antibodies13-15 that

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specifically interact with GM event biomarkers for GMO assay. Among them, the

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protein-based immunoassays, such as ELISA16,17 and immunochromatographic test

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strip,18 display concrete advantages in simple manipulation, no need of sophisticated

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instruments and low cost. Especially, the immunochromatographic test strip has the

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superiority of fitting for field tests. Unfortunately, so far there are merely several

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protein-based methods reported for qualitative or quantitative detection of GMOs.

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The most reported methods usually adopted a visible colorimetric readout strategy,

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which always suffered from insufficient sensitivity. Further efforts are needed to

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develop a more sensitive immunoassay to expand the potential applications of the

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protein-based approach.

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In recent years, various immunoassays are developed with different detection

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methodologies, such as colorimetry,19 electrochemistry,20 fluorescence,21 surface

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plasmon

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(ECL).25-27 Because of their low background and excellent sensitivity, ECL

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immunoassay involving chemiluminescence produced by electrochemical reaction

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have attracted increasing attention.28-33 In the process of ECL detection, the

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luminescence signal for the transduction of recognition event occurring on the

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electrode is triggered by the redox-active substance in the absence of an excitation

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light source. Therefore, the ECL detection shows a lower background signal and an

resonance,22 photoelectrochemistry23,24 and electrochemiluminescence

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improved sensitivity for immunoassay.34 The most reported ECL immunoassays are

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based on labeling technology,35,36 which usually suffer from a labor-intensive and

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time-consuming labeling process and the complex assay procedure. However, a

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label-free strategy that relies on the change of output signal upon the biological

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binding event can be applied to ECL immunoassay to overcome the shortcomings

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mentioned above. This way, the label-free ECL immunoassay possesses an innate

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high sensitivity and simplicity with reduction of the complexity in signal acquisition.

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In this work, novel carbon nanoparticles (CNPs) derived from printer toner were

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inventively used as an ideal antibody carrier and an efficient electrode material due to

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its large surface area and ideal electrical conductivity. Luminol was adopted as ECL

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reagent due to its low oxidation potential yet high emission efficiency.37,38 GM

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rapeseed RF3 event is the earliest approved GM variety as raw materials of food or

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livestock feed in China. A label-free ECL immunoassay targeting PAT/bar protein was

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established with RF3 event as model by immobilizing the CNPs-conjugated antibody

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for PAT/bar protein (CNPs-anti-PAT) on a working electrode. PAT/bar protein which

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was encoded by the bar gene isolated from Streptomomyces viridochromogenes is one

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of the most common biomarkers in GM rape.

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Experimental

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Materials and equipment

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Printer toners used to prepare CNPs, PAT/bar protein and monoclonal antibody for

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PAT/bar protein were obtained from Riogene Inc (Shanghai, China). Bovine serum

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albumin (BSA) and cetyltrimethyl ammonium bromide (CTAB) were purchased from

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Biosharp Co., Ltd (Hefei, China). Luminol utilized as ECL reagent was provided by

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Sigma-Aldrich Chemical Co., Ltd (Saint Louis, USA). Trifluoroacetamide and

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glutaraldehyde were supplied by Sinopharm Chemical Reagent Co., Ltd (Shanghai,

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China). Chitosan (CS) was obtained from Aladdin Chemistry Co., Ltd (Shanghai,

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China). The CS solution at 1.0% was prepared by ultrasonically dissolving 250 mg of

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CS in 25 mL of 1.0% acetic acid. The PAT/bar protein was diluted with phosphate

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buffer (PB) at 0.010 M and pH 7.4 containing 1.0% BSA, and the other biomaterials

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were PB. The blocking buffer was PB containing 2.0% BSA and 0.050% Tween-20.

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All aqueous solutions were prepared with ultrapure water (18.2 MΩ) purified by a

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Millipore Milli-Q Acedemic System. All other reagents were of the best grade

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available and used without further purification.

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Genuine seeds of GM rapeseed RF3, T45, GT73 and GM rice event carrying a bar

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gene were collected and identified by Supervision and Test Center (Wuhan) for

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Environmental Safety of Genetically Modified Plants of the Chinese Ministry of

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Agriculture. GM rice TT51-1 seeds were kindly supplied by Huazhong Agricultural

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University. A GM soybean event carrying bar gene was kindly provided by Professor

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Zhihui Shan at Chinese Academy of Agricultural Sciences. Non-GM seeds were

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purchased from the local market.

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The ECL measurements were performed with a MPI-A ECL analyzer (Xi’an

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Remax Electronic Science & Technology Co., Ltd., Xi’an, China) equipped with a

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three-electrode system composed of a platinum wire counter electrode, an Ag/AgCl

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reference electrode saturated with 0.50 M KCl, and a glassy carbon working electrode

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(GCE) with a diameter of 3 mm. Electrochemical impedance spectroscopy (EIS) was

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conducted by an Autolab PGSTAT302 electrochemical workstation (Metrohm

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Autolab Co., Ltd., Herisau, Switzerland). The X-ray photoelectron spectroscopy (XPS)

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was measured with an Escalab 250Xi X-ray photoelectron spectroscope (Thermo

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Fisher Scientific, Waltham, USA). Cyclic voltammetry (CV) measurements were

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carried out using a Corrtest CS350H electrochemical workstation (Corrtest

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instruments Corp., Ltd., Wuhan, China). Scanning electron microscopy (SEM) images

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were obtained utilizing a VEGA 3 LMU scanning electron microscope (Tescan Ltd.,

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Brno-Kohoutovice, Czech Republic). Transmission electron microscopy (TEM) was

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examined on a JEM-2100 transmission electron microscope (Jeol Ltd., Tokyo, Japan).

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Preparation of genuine samples

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GM rapeseed RF3 and non-GM rape seeds were ground utilizing a blender (Midea

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type WBL25B26, Foshan, China). Standard samples for GM rapeseed quantification

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were prepared by mixing rapeseed RF3 powder with non-GM seed powder based on

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different mass ratios of 0.050, 0.10, 0.20, 0.50 and 1.0% GMOs, respectively.

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Subsequently, 10 g of the prepared standard sample was dispersed in 25 mL of

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ultra-pure water, followed by shaking for 10 min. After centrifugation at 8000 rpm

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for 10 min to remove the precipitate, the extracted solution was stored at 4 oC for

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further use.

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Preparation of amino-functionalized CNPs and CNPs-anti-PAT conjugate

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To prepare amino-functionalized CNPs, 10 g of printer toner was dispersed in 50

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mL of ultra-pure water, followed by centrifugation at 12,000 rpm for 30 min to

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discard the precipitate. Then 0.050 g of CTAB used as a protectant against

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nanoparticles aggregation was added into the above supernatant, followed by a

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sonication for 3 h to prepare the CNPs dispersion. Afterward, the obtained CNPs were

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treated with silanization. Typically, 10 mL of anhydrous ethanol and 1.0 mL of

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trifluoroacetamide were sequentially added into the dispersion under constant stirring

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at a rate of 200 rev/min, followed by reaction at 55 oC for 8 h under ventilation with

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nitrogen. The resulted mixture was centrifugally washed thrice at 12,000 rpm for 10

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min using anhydrous ethanol and 0.020 M PB at pH 7.4 in turn. The obtained

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amino-functionalized CNPs were re-dispersed in 50 mL of 0.020 M PB at pH 7.4.

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For preparation of the CNPs-anti-PAT conjugate, 1.0 mL of the functionalized

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CNPs solution was thoroughly mixed with 5.0 µL of glutaraldehyde, followed by

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addition of 100 µL of 0.10 mg/mL anti-PAT monoclonal antibody dissolved in 0.020

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M PB at pH 7.4. The mixture was stirred for 2.5 h at 4 oC, and then dialyzed against a

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buffer to remove excess glutaraldehyde. Finally, the resultant conjugate was stored at

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4 oC for further use.

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Preparation of the immunosensor

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The GCE was polished with 0.050-µm alumina powder, and then ultrasonically

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washed in 75% ethanol and ultra-pure water for 5 min in turn. Five microliters of

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immunosensing composite prepared by mixing the CS solution and the

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CNPs-anti-PAT conjugate at a ratio of 1:1 (v/v) was then deposited onto the surface 8


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of the working electrode. After the modified electrode was dried at room temperature

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(RT) to form film, it was blocked with 2.0% BSA for 40 min at RT to minimize the

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nonspecific adsorption. The resultant ECL immunosensor was rinsed with PB buffer

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and then ready for use.

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Assay procedure

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In order to perform a label-free ECL immunoassay, 30 µL of PAT/bar protein or

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GM rapeseed RF3 sample was dropped on the fabricated immunosensor, followed by

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incubation at 37 °C for 1 h. After a thorough washing with a PB buffer, the GCE was

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inserted into an electrochemical cell filled with 8.0 mL of 0.050 M KOH containing

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0.60 mM luminol. Finally, the ECL signal was triggered by the application of a cyclic

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voltammetry in the sweeping range from -0.3 V to 0.9 V, and collected by the ECL

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analyzer for quantification.

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Results and discussion

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Characterization of ECL immunosensor

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The CNPs were originally synthesized based on the usage of a simple and

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improved approach. The TEM image (Figure 1A) of the as-prepared CNPs showed

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that they were spherical particles with a mean diameter of 20 nm, which regularly

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led to a large surface area.39 Only a handful of ECL immunoassays involving carbon

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nanospheres identically prepared from glucose under high-heat conditions were

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developed to date.40 Therefore the simply synthesized CNPs derived from printer

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toner in presented work expands the application of carbon nanomaterials in 9



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biosensor.

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To further investigate the surface chemical composition of CNPs, XPS spectrum

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of the nanomaterial was measured. According to Figure S5, the main elements on the

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surface of the synthesized CNPs were C, O, N, and Si.

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SEM was used to characterize bare, CNPs doped CS membrane modified and

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CNPs-anti-PAT doped CS modified working electrodes. As presented in Figure 1B,

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the morphology of the bare electrode was uneven and inhomogeneous, which

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resulted in a poorly reproducible response. Compared with the bare sensor surface, a

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three-dimensional porous structure with uniform pore size was observed after CNPs

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were mixed with CS (Figure 1C), which resulted in an improved electrical

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conductivity and an increased loading capacity of the biomolecules.41 As shown in

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Figure 1D, as the CNPs-anti-PAT was doped in the CS film, the abundant antibodies

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were observed to cover the porous structure, generating a sensitive ECL response.

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EIS that obtained in 5.0 mM K3[Fe(CN6)]/K4[Fe(CN6)] solution containing 0.10

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M KCl was employed to acquire the charge transfer resistance (Ret ) of the GCE. As

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seen in the Figure 2A, the CS film deposited electrode showed the Ret of 523 Ω

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(curve a). A decrease in the Ret value (241 Ω, curve b) was obtained after the CNPs

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were doped into the CS membrane, indicating that the CNPs possessed high

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electrical conductivity to enhance the interfacial electron transfer. After the

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deposition of the CNPs-anti-PAT and BSA blocking, the Ret values increased from

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1459 Ω to 2235 Ω (curves c and d) since antibodies and BSA inhibited the electron

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transfer. A larger Ret value of 3720 Ω (curve e) was observed after incubating with

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the GM rapeseed RF3 sample because of the immunoreaction of antigen-antibody

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occurred on the electrode surface. Meanwhile, CV in K3[Fe(CN6)]/K4[Fe(CN6)]

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solution was measured to monitor the fabrication process of developed

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immunosensor. As presented in Figure S6, after the immobilization of CNPs (curve

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2), the redox peak currents became more visible than that of bare GCE (curve 1),

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which is attributed to the accelerated electron transfer activity of CNPs. The current

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intensities decreased progressively after the immobilization of CNPs-anti-PAT (curve

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3), BSA (curve 4) and GM rapeseed RF3 sample (curve 5) due to the hindrance of

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electron transfer by proteins on electrode surface. The results of Ret and CV tests

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demonstrated the successful assembly of the immunosensor. Thus the GMO

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detection could be achieved by the proposed ECL immunoassay.

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Principle of the proposed ECL immunoassay

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The principle of the proposed label-free ECL immunoassay is illustrated in Figure 3.

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In this protocol, The prepared CNPs possessing large surface and high electrical

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conductivity were applied to load huge quantities of antibodies though the steps of

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silanization and glutaraldehyde cross-linker. The CNPs-anti-PAT conjugate was

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further used to modify the electrode and develop the highly sensitive ECL

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immunoassay. When a PAT/bar protein was present in the sample solution, it could

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bind to the immobilized antibody on the sensor bioreceptor region to form

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antigen-antibody complex. This way the electron transfer between the electrode

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surface and the redox-active substance was hindered by this immunocomplex, leading

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to a decrease of ECL intensity as PAT/bar protein level increased. Consequently,

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PAT/bar protein detection was achieved just by monitoring the decrease of ECL

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signal of the developed ECL immunoassay.

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Optimization of detection conditions

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The ECL response of the proposed immunoassay was influenced by the amount of

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antibodies and reaction conditions. As shown in Figure S1, the dependence of an ECL

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response on the concentration of the CNPs-anti-PAT conjugate was investigated

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utilizing RF3 standard sample at the mass fraction of 0.10% and non-GM rapeseed

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sample (as a blank) in parallel. It was found that the signal-to-blank ratio reached the

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minimum at the dilution time of 1:10, suggesting a strong inhibition of ECL signal.

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ECL responses in Figure S4 decreased with the increase of incubation time and

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reached the plateau after 60 min, implying that the immuno-binding reached the

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saturation. Therefore, the CNPs-anti-PAT conjugate at the dilution time of 1:10 and

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the incubation time of 60 min was routinely applied for the further investigation.

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Because luminol showed a strong and stable ECL in an alkaline medium, while it

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was non-luminous in neutral and acid ones, the key factors influencing the ECL

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response were the concentrations of luminol solution and alkaline medium. Figure S2

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exhibits the effect of the concentration of luminol on the response ratio between RF3

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standard sample at 0.10% and non-GM rapeseed sample. The result showed the

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signal-to-blank ratio that was minimum when the concentration of luminol was 0.60

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mM, implying that the ECL signal inhibition was strongest. In this study, a KOH

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solution was used as an alkaline medium to dissolve luminol for the purpose of

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acquiring high ECL efficiency. As seen in Figure S3, the ECL response was 12


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accelerated with the concentration of KOH solution. While the KOH concentration

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was higher than 0.050 M, the ECL intensity began to decrease, indicating that the high

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alkaline solution inhibited the oxidation of luminol. Consequently, a 0.050M KOH

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solution containing 0.60 mM luminol was adopted as the ECL substrate.

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Specificity, repeatability and stability of ECL immunoassay

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In order to estimate the specificity of the proposed method for PAT/bar protein

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detection, the interferents of various GM event proteins which usually are present in

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GM crops were investigated. TT51-1 rice powder (Cry 1Ab/Ac protein), GT73

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rapeseed powder (CP4-EPSPS protein) and T45 rapeseed powder (PAT/pat protein)

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dispersed in the blank solution were utilized as the interfering proteins. A blank

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signal was obtained from a non-GM rapeseed solution. Figure 2B presents the ECL

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responses from the disruptors with the mass fraction of 0.10%. All responses showed

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slight changes in comparison with the blank signal. While a significant decrease of

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ECL emission of luminol was observed from the rapeseed RF3 sample at the same

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concentration. The following equation was used to calculate the degree of

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interference (DI) value of these interfering proteins:

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DI = A − C × 100% B−C

(1)

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where A, B and C represent the ECL responses from the interfering proteins, RF3

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sample and the blank, respectively. The DI values for Cry 1Ab/Ac protein,

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CP4-EPSPS protein and PAT/pat protein were 3.3%, 4.2% and 4.5%, respectively.

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Besides, mixture 1 composed of RF3 sample and the three interferents (all at 0.10%),

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and mixture 2 composed of the three interferents (all at 0.10%) were prepared and

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assayed with the developed ECL immunoassay. The DI value for mixture 2 was

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calculated to be 4.8%. Furthermore, only a negligible difference of 1.7% was

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observed from the ECL response of mixture 1 in comparison with that of RF3

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sample. Therefore, the developed method was not prone to be interfered by these

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common GM event proteins, thus exhibiting ideal specificity for PAT/bar protein

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assay in a real sample.

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In addition, to investigate the repeatability of the proposed method, three GM

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rapeseed RF3 samples at different contents (0.10%, 0.50% and 1.0%) were

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repeatedly assayed for 5 times at five different time points (Figure 2C). The relative

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standard deviations (RSDs) were 3.7%, 5.0% and 6.3%, respectively. Furthermore,

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the storage stability of the developed ECL immunoassay was also evaluated. It still

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remained 91.3% of the initial response after storage at 4 oC for two weeks. The

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operational stability of the proposed approach was studied by measuring the ECL

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emission of the luminol system under continuous cyclic voltammetry for 10 cycles

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(Figure 2D). The stable ECL signals were observed with a RSD of 1.7%. Therefore,

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the repeatability and stability of the developed method was acceptable.

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Analytical performance for PAT/bar protein and GM rapeseed RF3

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Under the optimum conditions, the ECL response linearly decreased with the

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increase of PAT/bar protein concentration due to the inhibition of electron transfer

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resulting from generation of antigen-antibody complex. As shown in Figure 4A, a

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dose-response calibration curve for PAT/bar protein detection was observed in the 14


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concentration range of 0.10 - 10 ng/mL. The linear regression equation was

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expressed as I (a. u.) = -170 C (ng/mL) + 4256 (I and C represent the ECL intensity

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and PAT/bar protein concentration, respectively), with the correlation coefficients of

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0.9960. The limit of detection (LOD) defined as the sample content generating a

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signal to noise ratio of 3 42,43 for PAT/bar protein was 0.050 ng/mL.

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Hereafter, the proposed ECL immunoassay targeting PAT/bar protein was applied

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to detect the content of GM rapeseed RF3 in samples. As shown in Figure 4B, the

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ECL responses of RF3 samples exhibited a similar trend to that of PAT/bar protein,

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and linearly decreased with increasing GM rapeseed RF3 content ranging from

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0.050% to 1.0%. The linear regression equation was I (a. u.) = -2337 C (%) + 3113 (I

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and C denote the ECL intensity and GM rapeseed RF3 content, respectively), with

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the correlation coefficients of 0.9949. The developed method could determine the

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content of GM rapeseed RF3 down to 0.020% (S/N = 3). Compared to the published

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approaches for a bar gene target detection, such as the PCR screening method,44,45

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DNA chip,46 ELISA 47 and protein test strip,48 this method displayed comparable or

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even higher detection sensitivity. Moreover, the whole assay process can be

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accomplished within 65 min by utilizing a ready-for-use immunosensor that had

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already been modified and blocked, Therefore, the developed method can be used

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for rapid detection and quantification of GMO content in test samples. In

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comparison with other biosensors for GMO detection including aptamer-based

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aptasensor,49 quartz crystal microbalance biosensor

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biosensor,51 the presented approach does not require complicated nucleic acid

50

and surface plasma resonance

15



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extraction and sophisticated instruments, thereby facilitating the development of

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portable and facile device in further study.

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Application in genuine GM rapeseed samples

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Three unknown GM rapeseed samples carrying a bar gene were assayed for the

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purpose of validating the reliability of the proposed approach. After five

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measurements for each sample, the GMO contents in these genuine samples were

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measured to be 0.11%, 0.060% and 0.46%. Furthermore, known amounts of RF3

333

standards were spiked into the three samples to conduct the recovery tests. As seen in

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Table 1, the recoveries were all between 80.0% and 106.0%, and the RSDs were all

335

less than 8.0%, demonstrating acceptable reliability of the proposed method.

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Additionally more GM crops targeting bar gene at contents of 0.10%, 0.50% and

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1.0% including a GM rice event and a GM soybean event were detected using the

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proposed ECL immunoassay in Figure S7. ECL responses to contents in diverse

339

species of GMO have similar pattern, suggesting that ECL immunoassay in presented

340

study is not specie-specific and therefore applicable to analyse GMO containing

341

PAT/bar protein.

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In conclusion, an ECL immunoassay targeting the PAT/bar protein was designed

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with a very facile protocol and developed for the highly sensitive detection of

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PAT/bar protein in GM rapeseed. The PAT/bar protein could be assayed in a

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label-free mode based on the inhibition of electron transfer and redox-active

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substance diffusion resulting from the formation of an immunocomplex. The

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application of novel CNPs and ECL detection greatly improved the detection

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sensitivity of the proposed immunoassay, which was comparable to that of the highly 16


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PCR-based approach. The proposed ECL immunoassay showed good linearity

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between ECL intensity and GMO content or PAT/bar protein amount. The

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quantification of GM rapeseed in samples demonstrated that this method can rapidly

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measure the GMO content in samples by utilizing the serially diluted GM reference

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materials as calibrants. With PAT/bar protein as calibrants this approach can be also

354

used to accurately quantify the expression level of bar gene in transformation. The

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obtained data would contribute to screen eminent transformation in genetic

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engineering research. The developed protocol possesses the advantages of simple

357

procedure, time-saving analysis and high sensitivity, providing a powerfully

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alternative tool for detection of GMOs and evaluation of exogenous protein

359

expression level in GMOs. In the future work, we will attempt to develop an ECL

360

immunoassay chip for high-throughput determination of GM crops based on the

361

obtained results, further improving the detection efficiency.

362

Acknowledgement

363

This work was supported by the National Grand Project of Science and Technology

364

(2018ZX08012001-005) and the Natural Science Foundation of China (31271880 and

365

31671733).

366

Competing interests

367 368

The authors declare that they have no competing interests. Supplementary Materials

17



369

Effect of the dilution time of CNPs-anti-PAT on the signal-to-blank ratio, effect of

370

the concentration of luminol on the signal-to-blank ratio and effect of the

371

concentration of KOH solution on the ECL response.

372 373

18


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374 375 376 377 378

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26


Page 26 of 37

Page 27 of 37


546

Captions for Figures

547

Figure 1. (A) TEM image of CNPs. SEM images of (B) bare, (C) CNPs doped CS

548

membrane modified and (D) CNPs-anti-PAT doped CS modified working

549

electrode.

550

Figure 2. (A) EIS spectra of electrodes modified with (a) CS, (b) CNPs/CS, (c)

551

CNPs-anti-PAT/CS,

552

protein/BSA/CNPs-anti-PAT/CS.

553

immunoassay. (C) ECL responses of the developed method for GM rapeseed RF3

554

sample at 0.10%, 0.50% and 1.0%, respectively. (D) ECL emission under 10 cycles

555

of continuous potential scans for the detection of GM rapeseed RF3 sample at

556

0.10%.

557

Figure 3. Schematic illustration of the proposed ECL immunoassay for PAT/bar

558

protein detection with a label-free mode.

559

Figure 4. ECL responses of (A) PAT/bar protein at the concentrations of 0, 0.10,

560

0.50, 1.0, 5.0, and 10 ng/mL, from top to bottom; and (B) GM rapeseed RF3 at the

561

mass fractions of 0, 0.050%, 0.10%, 0.20%, 0.50% and 1.0%, from top to bottom.

562

Inset: Calibration curves, where n = 5 for each point.

(d)

BSA/CNPs-anti-PAT/CS (B)

Specificity

27


of

and the

PAT/bar

proposed

ECL


563

Figure 1

564

28


Page 28 of 37

Page 29 of 37


565

Figure 2

566

29



567

Figure 3

568

30


Page 30 of 37

Page 31 of 37


569

Figure 4

570 571

31



Page 32 of 37

Table 1 Recoveries of GM rapeseed RF3 spiked in the genuine GM rapeseed samples obtained by the proposed ECL immunoassay. Samples number

1

2

3

Initial (%)

0.11

0.060

0.46

Added (%)

0.10

0.50

0.10

0.50

0.10

0.50

Found (%) RSD (%) Recovery (%)

0.19 7.8 80.0

0.64 7.7 106.0

0.15 4.9 90.0

0.50 5.4 88.0

0.55 6.7 90.0

0.97 8.0 102.0

32


Page 33 of 37


33x37mm (300 x 300 DPI)



27x19mm (600 x 600 DPI)


Page 34 of 37

Page 35 of 37


24x20mm (600 x 600 DPI)



18x6mm (600 x 600 DPI)


Page 36 of 37

Page 37 of 37


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