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A simple and sensitive competitive bio-barcode immunoassay for triazophos based on multi-modified gold nanoparticles and fluorescent signal amplification
Accepted Manuscript A simple and sensitive competitive bio-barcode immunoassay for triazophos based on multi-modified gold nanoparticles and fluorescent signal amplification Chan Zhang, Pengfei Du, Zejun Jiang, Maojun Jin, Ge Chen, Xiaolin Cao, Xueyan Cui, Yudan Zhang, Ruixing Li, A.M. Abd El-Aty, Jing Wang PII:
S0003-2670(17)31209-6
DOI:
10.1016/j.aca.2017.10.032
Reference:
ACA 235506
To appear in:
Analytica Chimica Acta
Received Date: 21 August 2017 Revised Date:
20 October 2017
Accepted Date: 26 October 2017
Please cite this article as: C. Zhang, P. Du, Z. Jiang, M. Jin, G. Chen, X. Cao, X. Cui, Y. Zhang, R. Li, A.M. Abd El-Aty, J. Wang, A simple and sensitive competitive bio-barcode immunoassay for triazophos based on multi-modified gold nanoparticles and fluorescent signal amplification, Analytica Chimica Acta (2017), doi: 10.1016/j.aca.2017.10.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ms. No.: ACA-17-1940
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A simple and sensitive competitive bio-barcode immunoassay for triazophos based on
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multi-modified gold nanoparticles and fluorescent signal amplification
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Chan Zhanga, Pengfei Dub, Zejun Jianga, Maojun Jina, *, Ge Chena, Xiaolin Caoa, Xueyan
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Cuia, Yudan Zhanga, Ruixing Lia, A. M. Abd El-Atyc, d, Jing Wanga, *.
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a
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Agro-Product Quality and Safety, Chinese Academy of Agricultural Sciences; Key Laboratory
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of Agro-Product Quality and Safety, Ministry of Agriculture, Beijing 100081, P. R. China
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Institute of Quality Standard and Testing Technology for Agro-Products, Key Laboratory of
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b
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Key Laboratory od Agro-Products Processing Technology of Shandong Province; Key
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Laboratory of Novel Food Resources Processing, Ministry of Agriculture, 202 Gongye North
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Road, Jinan 250100, P.R. China
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c
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Giza, Egypt
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d
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Konkuk University, Seoul 143-701, Republic of Korea
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Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences;
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Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211
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Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine,
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* Corresponding authors
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Tel.:
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+86-10-8210-6568; Fax: +86-10-8210-6567. E-mail: [email protected] (J. Wang)
+86-10-8210-6570.
E-mail:
[email protected]
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1
(M.
J.
Jin)
and
Tel.:
ACCEPTED MANUSCRIPT Abstract
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A simple and highly sensitive immunoassay based on a competitive binding and bio-barcode
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amplification was designed for detection of small molecules, triazophos. The gold
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nanoparticles (AuNPs) were modified with monoclonal antibodies and 6-carboxyfluorescein
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labeled single-stranded thiol-oligonucleotides (6-FAM-SH-ssDNAs); the fluorescence of
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6-FAM was quenched by AuNPs. Ovalbumin-linked haptens were coated on the bottom of
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microplate to compete with the triazophos in the sample for binding to the antibodies on the
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AuNP probes. The fluorescence intensity was inversely proportional to analyte concentration.
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Parameters of AuNP probes preparation and immune reaction were optimized. At the optimal
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conditions, the salting process was shortened to 1 h and 166 ± 9 ssDNAs were loaded onto a
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single AuNP. The competitive fluorescence bio-barcode immunoassay was performed on
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water, rice, cucumber, cabbage and apple samples. The linear range of the method was 0.01–
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20 µg L-1and the limit of detection (LOD) was 6 ng L-1. The recovery and relative standard
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deviations (RSDs) ranged from 85.0–110.3% and 9.4–17.4%, respectively. Good correlations
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were
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chromatography-tandem mass spectrometry (LC-MS/MS). In conclusion, it is suggested that
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the competitive fluorescent bio-barcode immunoassay had the potential to be used as a
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sensitive method for detection of a variety of small molecules in various samples.
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Keywords Immunoassay; Fluorescence; Bio-barcode; Gold nanoparticles; Triazophos.
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the
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1. Introduction Triazophos (O, O-diethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) phosphorothioate) is a
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ACCEPTED MANUSCRIPT broad-spectrum, moderately toxic insecticide that has been widely used on a variety of crops
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in recent years as a good alternative to highly toxic organophosphates such as parathion,
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parathion methyl, and methamidophos. However, it has a relatively high stability, and its long
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half-life presents potential risks to human health and the environment [1, 2]. There is
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widespread concern over the presence of triazophos residues in environment as well as foods
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[3, 4]. Traditional analytical methods for determination of triazophos include gas
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chromatography (GC) and liquid or gas chromatography coupled with mass spectrometry
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(MS). While these methods are sensitive and accurate, they require expensive equipments,
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skilled technicians, and lengthy analysis [5-8]. In numerous instances, immunoassays have
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proven to be exceptional tools for pesticide detection, as they are simple, cost-effective, and
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rapid. Among immunoassay types, enzyme linked immunosorbent assay (ELISA) is widely
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popular; however, its sensitivity does not meet testing requirements in some cases. Thence,
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the establishment of detection methods with high sensitivity has stimulated extensive
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research.
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Gold nanoparticles (AuNPs) are widely used in the detection and analysis of chemical and
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biological molecules, owing to their unique optical, catalytic, electrical, and chemical
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properties [9-11]. Huo’s group [12] first measured the specific interactions between protein
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A-immobilized AuNPs and human IgG via detecting the average particle size change using
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dynamic light scattering (DLS). Storhoff et al. [13] detected DNA sequences based on the
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colorimetric scattering of AuNP probes. Hou et al. [14] designed an impedimetric
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immunoassay protocol for carcinoembryonic antigen detection based on enzyme-triggered
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formation of tyramine-enzyme repeats on AuNP. In 2003, the bio-barcode technique,
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molecules, was first developed by the Mirkin group to detect and quantify prostate-specific
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antigen [15]. Subsequently, this technique was applied to DNA detection [16, 17]. The system
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relies on the use of magnetic microparticle probes (MMPs) functionalized with antibodies as
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well as AuNPs encoded with bio-barcodes and antibodies. The targets, which are dissolved in
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test solution, are captured by the two probes to form a sandwich construction. The
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bio-barcodes released from the AuNPs are initially determined by silver amplification or
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polymerase chain reaction (PCR). The combination of molecular biology and labeling
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techniques is a hotspot in the detection technology [18-20]. Researchers have modified the
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detection strategy of bio-barcode immunoassay by enzymes, RCA reaction or other schemes
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based on. Liu et al. [21] detected proteins using enzyme-labeled AuNP probes that were
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coated with antibody, single-stranded DNA (ssDNA), and horseradish peroxidase (HRP). Yan
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et al. [22] developed an amplification strategy based on rolling circle amplification (RCA)
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reactions. During the RCA reaction, biotin labels were incorporated into the RCA-generated
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long ssDNA, which bound to avidin-HRP (Av-HRP) to produce enzymatic catalysis-based
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colorimetric signals. Lv et al. [23] modified AuNPs with antibody and G-quadruplex/hemin.
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The hemin released by Exonuclease I can capture the photogenerated electrons to
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significantly amplify the signal intensity. Zhou et al. [24] exploited plasmid-encoded peptide
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tags as surrogate molecules for the matrix-assisted laser desorption/ionization time-of-flight
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MS identification of target DNA.
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It has to be noted that the sandwich construction was not suitable for pesticides and other
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small-molecule haptens, as the latter possess only one antibody-combining site. To conquer
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sandwich construction. In 2015, our group first developed a competitive bio-barcode
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amplification immunoassay for the detection of pesticide residue using quantitative reverse
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transcription (RT)-PCR [25]. This method had a high sensitivity but was time-consuming,
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labor-intensive, and expensive. Afterward, we switched our strategy to a sensitive and
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relatively simple method, in which the AuNPs were modified by antibodies and HRP-labeled
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ssDNA. After magnetic separation, the substrate was added and the absorbance produced by
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the enzyme-catalyzed colorimetric reaction was measured. [26].
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Herein, to further simplify the technique and improve the sensitivity of the previously
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developed bio-barcode immunoassay, we designed a method for triazophos detection using
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only one type of probe. The AuNPs were modified with mAb and 6-FAM-SH-ssDNAs and
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the fluorescence of 6-FAM was quenched by AuNPs. After the competitive reaction between
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ovalbumin-linked
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hapten-mAb-AuNP probe complexes were formed at the bottom of the microplate, as shown
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in Scheme 1. Dithiothreitol (DTT) was used to replace the 6-FAM-SH-ssDNA on the
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complex and the released fluorescence signal was detected.
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2. Experimental section 2.1 Chemicals and materials
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The standard for triazophos (98%) and structural analogues (>95%), bovine serum albumin
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(BSA), goat anti-mouse IgG, polyethylene glycol (PEG) 20000, DTT, Tris-EDTA (TE) buffer
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solution (pH 7.4), hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O, > 99.9%), and
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ACCEPTED MANUSCRIPT trisodium citrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). The triazophos
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hapten and monoclonal antibodies (mAbs) from mouse ascites were generously gifted by the
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Institute of Pesticide and Environmental Toxicology (IPET), Zhejiang University, China.
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Oligonucleotides (3’-FAM, 5’-SH C6) were generated by Shanghai Sangon Biotechnology Co.
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Ltd (Shanghai, China). The 3, 3’ 5, 5’-Tetramethylbenzidine (TMB) ELISA Substrate was
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purchased from TransGen Biotech Co., Ltd (Beijing, China). Octadecylsilyl (C18, 40–60 µm),
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and primary secondary amine (PSA, 40–60 µm) were obtained from Agela (Tianjin, China).
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LC-grade acetonitrile and methanol were supplied by Fisher Scientific (Pittsburgh, PA, USA).
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All other chemicals, such as anhydrous magnesium sulfate (MgSO4) and sodium acetate
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(NaCl) and organic solvents of analytical grade or higher were purchased from Beijing
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Chemical Industry Group Co., Ltd (Beijing, China). Ninety-six-well plates (transparent and
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black, flat bottom) were obtained from Corning, Inc. (Corning, NY, USA). Ultrapure water (≥
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18.2 MΩ·cm) produced by a Milli-Q water purification system (Millipore, Bedford, MA,
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USA) was used in all e experimental works.
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The buffers used in this study were as follows: salting buffer (consisting of 100 mM
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phosphate buffered saline (PBS) and 1.3 M NaCl); blocking and storage buffer (consisting of
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10 mM PBS (pH 7.4), 1% BSA, and 1% PEG 20000); coating buffer (consisting of 50 mM
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carbonate buffer solution (CBS, pH 9.6)); washing buffer (PBST) (consisting of 10 mM PBS
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(pH 7.4) and 0.05% Tween 20); blocking buffer (consisting of 10 mM PBS (pH 7.4) and 2%
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BSA); assay buffer (consisting of 10 mM PBS (pH 7.4) and 0.05% BSA); and ligand
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exchange buffer (comprising 10 mM PBS (pH 8.0)).
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2.2 AuNPs synthesis and modification The 13 nm AuNPs were prepared as previously described [27]. Briefly, 100 mL of 1 mM
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HAuCL4 aqueous solution was heated to boiling with stirring and refluxing, followed by the
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addition of 50 mL of 38.8 mM trisodium citrate rapidly to the boiling solution. The color
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changed from pale yellow to deep red within 5 min; the solution was boiled and stirred for an
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additional 15 min. After the solution was cooled to ambient temperature (25°C), it was
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filtered through a 0.22-µm cellulose nitrate filter. The prepared colloidal particles were
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characterized by UV-Vis spectroscopy (Infinite M200 PRO microplate reader, TECAN,
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Switzerland) and transmission electron microscopy (TEM, TEI Technai G2 F20, Thermo
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Fisher Scientific, MA, USA).
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AuNP probes modified with mAbs and 6-FAM-SH-ssDNA were prepared following a
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previously reported protocol, with minor modifications [16]. The pH value of the AuNP
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solution (1 mL) was adjusted to 9.0 with 30 µL 0.1 M K2CO3, and 18.12 µg mAb was
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thoroughly mixed into the colloidal solution. After withstanding for 1 h, 2 nmol of freshly
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reduced thiolated ssDNA was mixed into the AuNP-antibody complex solution. Subsequently,
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30% PEG 20000 aqueous solution and salting buffer were added to final concentrations of 1%
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and 0.137 M, respectively, followed by incubation at room temperature for 2 h. Thereafter,
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BSA was added to a final concentration of 1% to block the bare surfaces, and the mixture was
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incubated for another 1 h. Finally, the unbound antibody and oligonucleotides were removed
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by two centrifugation steps at 15000 rpm for 20 min at 4°C (Thermo Fisher Scientific, MA,
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USA). The red precipitate was suspended in 200 µL of blocking and storage buffer and could
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stay stable for at least two weeks.
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2.3 Competitive fluorescence bio-barcode amplification immunoassay First, 100 µL per well of OVA-triazophos hapten in CBS was added to a black polystyrene
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microplate and incubated overnight at 4°C. The coated plate was washed three times with
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PBST followed by blocking with 300 µL blocking buffer for 1 h at 37°C. Then 50 µL of
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sample extract or triazophos standard solution, in 10% (v/v) methanol-PBS (0.01 M, pH 7.4),
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and 50 µL AuNP probes, diluted in assay buffer, were sequentially added to the microplate.
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During incubation at 37°C for 1 h, the hapten coating the bottom of the microplate and the
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triazophos in the sample compete for binding with the mAb adsorbed on the surface of the
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AuNPs. When bound, the fluorescence of 6-FAM is quenched by the AuNPs due to
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fluorescence resonance energy transfer (FRET) [28-31]. The unbound pesticide and AuNP
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probes were removed by three washes with PBST. Finally, 100 µL of DTT was added to
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displace the 6-FAM-SH-ssDNA by ligand exchange [16]. The inhibition of fluorescence
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intensity (Ex489nm/Em521nm, Infinite M200 PRO microplate reader, TECAN, Switzerland) and
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the logarithm of the pesticide concentration exhibited a linear relationship.
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2.4 Indirect competitive ELISA
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The coating, blocking, and washing of 96-well transparent polystyrene microplates were
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performed as described above.
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standard solution in 10% (v/v) methanol-PBS (0.01 M, pH 7.4) and 50 µL of diluted mAb
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were sequentially added to the microplate. After incubation at 37°C for 1 h and three washes
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with PBST, 100 µL goat anti-mouse IgG was added, and the plate was incubated at 37°C for
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1 h. Afterward, 100 µL TME was added and the plate was incubated for 15 min at 37°C.
Subsequently, 50 µL of sample extract or triazophos
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Finally, 50 µL of 2 M H2SO4 was added to terminate the reaction and the absorbance at 450
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nm was measured.
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2.5 Sample treatment The samples were tap water from the laboratory, and rice, cucumbers, cabbages, and apples
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bought from a local market in Beijing. All samples were proved to be free from triazophos
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using LC-MS/MS. The water samples were diluted directly with 0.01 M PBS and 10%
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methanol. The pretreatments of other samples were carried out according to the quick, easy,
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cheap, effective, rugged, and safe “QuEChERS” with slight modifications [32]. Briefly, 10 g
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(5 g for rice) samples were weighed into 50 mL centrifuge tubes and mixed thoroughly by
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vortexing for 1 min with 10 mL acetonitrile (5 mL of water was added to the rice samples
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before acetonitrile). After adding 4 g MgSO4 and 1 g NaCl, the mixture was vigorously
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shaken for 1 min and then centrifuged for 5 min at 5000 rpm. Afterward, 2 mL of supernatant
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was transferred to a 10 mL centrifuge tube containing 100 mg PSA, 100 mg C18 and 150 mg
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MgSO4. After vortexing for 1 min, the tubes were centrifuged for 5 min at 10000 rpm. Half of
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the supernatant was transferred to injection vials for LC-MS/MS detection (See Suppl.
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materials) and the other was concentrated under a stream of nitrogen. Finally, the residue was
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dissolved in 10% methanol-PBS and examined by competitive bio-barcode fluorescent
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amplification immunoassay and ELISA.
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3. Results and discussion
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3.1 AuNP probe preparation and optimization
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A solution of 13 nm AuNPs, which exhibited a maximum absorption at 518 nm (Fig. 1(A),
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ACCEPTED MANUSCRIPT black line) was prepared according to the citrate reduction method. The TEM image in Fig.
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1(B) demonstrates that the prepared AuNPs exhibited consistent diameter and did not
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aggregate. The concentration of prepared AuNPs calculated by the Beer-Lambert Law was
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9.9 nM, using an extinction coefficient of 2.47×108 M−1·cm−1 [33]. The gold nanoparticles
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were simultaneously functionalized with 6-FAM-SH-ssDNA and triazophos mAbs. A salt
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aging process is necessary to load the negatively charged DNA onto the citrate ion-adsorbed
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surface of AuNPs. To prevent the irreversible aggregation of colloidal gold, the stepwise
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addition of NaCl often takes one to two days. A previous study indicated that high molecular
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weight PEG stabilizes the system by depletion stabilization, and over 700 DNAs may be
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loaded onto each 50 nm AuNP with 600 mM NaCl within 2 h [34]. Therefore, this experiment
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tested the stability of antibody-modified AuNPs in PEG 20000 by NaCl titration [16]. As
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shown in Fig. A.1 (A), the maximum absorption wavelength displayed a redshift in PEG
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20000 concentrations below 1%, but remained at 524 nm in higher concentrations.
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Comparative tests with the traditional, time-consuming method revealed that PEG 20000
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reduced the irreversible aggregation and the occurrence of oil membrane (Fig. A. 1 (B)). The
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presence of PEG 20000 made the time-consuming salting process unnecessary. The
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6-FAM-SH-ssDNA was maximally loaded after 1 h. It has to be noted that each
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centrifugation step may cause a loss of approximately 10% of AuNPs, and approximately 96%
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supernatant could be removed. Thus, to avoid excessive loss of gold colloidal, two times
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centrifugation are enough to remove most of the unbound materials.
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Previous studies have demonstrated that the nucleobase sequence plays a role in the binding
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of thiol-oligonucleotides to AuNPs, because of the different adsorption affinity of ssDNA
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AuNP surface compared to guanine, cytosine, and alanine deoxyribonucleotides (dG, dC, and
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dA, respectively), which contributes to substantially higher surface coverage of strands with
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(dT)20 than (dA)20 [35,36]. In addition, adsorption of deoxynucleosides, especially dA to gold
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causes the ssDNA strands to lay flat, therefore occupying more surface area than upright
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strands would, and hindering subsequent thiol-ssDNA adsorption [37]. Therefore, to select
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the optimal strand for the probes, various sequences and lengths of ssDNA were used to label
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the AuNP surface, as shown in Table 1. Sequences 1-6 were used to test the sequence effect
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on the coverage and sequences 6-10 were used to optimize the sequence length. The
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fluorescence intensity markedly increased when the spacer increased from (dT)0 to (dT)5, then
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increased slowly as the dT spacer length increased from 5 to 25 bases (Fig. 2(A)). Moreover,
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the fluorescence intensity and ssDNA coverage substantially decreased as ligand length
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increased from 5 to 25 bases (Fig. 2(B)). The number of ssDNA molecules coating each
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particle was calculated by detecting the fluorescence intensity of the supernatant and
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quantifying it based on a standard curve of fluorescence (Fig. A.2) [31]. For a gold solution
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without PEG 20000, long oligonucleotides enhance the surface coverage and stability of
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AuNPs due to steric hindrance [35]. However, interestingly, in the presence of PEG, the
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shorter the oligonucleotides were, the higher the coverage was, since shorter oligonucleotides
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have smaller steric hindrance. Thence, an oligonucleotide sequence of (dT)5 was used to
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prepare the AuNP probes.
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The mAb and thiol-oligonucleotide coverage affects the sensitivity of the method. The
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concentrations of mAb and ssDNA were therefore optimized. Firstly, AuNP probes were
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concentrations of 9.0, 18.1, 36.2, and 72.5 mg L -1 with excess ssDNA (molar rate, 500:1).
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The fluorescence intensity of the supernatant before and after modification was measured and
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the difference represented the amount of ssDNA loaded on the particle surface. Because of
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the increase of surface occupancy by mAbs, the fluorescence intensity decreased gradually
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when the mAbs concentration increased. However, when the probes were added to the
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microplate to combine with the coated triazophos hapten, the maximum fluorescence
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intensity appeared at 36.2 mg L -1. The lower fluorescence value of probes prepared with
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mAb concentrations of 9.0 and 18.1 mg L -1 might be due to the decreased antibodies on the
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particle surface (to form probe-antibody-hapten complexes), despite these probes carrying
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more 6-FAM-SH-ssDNA. Otherwise, the concentration of ssDNAs was optimized by adding
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a series amount of ssDNA to the final molar ratio of ssDNA and AuNPs of 100:1, 200:1,
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300:1, 400:1 and 500:1. As the results shown, the fluorescence increased accordingly as the
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ratio increased from 1:100 to 1:300, and showed a decrease trend as the ratio continued to
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increase to 1:500. Therefore, 18.1 mg L
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6-FAM-SH-ssDNA/AuNPs was used in subsequent experiments.
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3.2 Characterization of AuNP probes
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As shown in Scheme 1, the AuNP probes were modified with triazophos mAbs and
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6-FAM-SH-ssDNA using the optimized conditions and characterized by UV-Vis
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spectroscopy and TEM. Fig. 1(A) shows that after modification with mAbs and
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6-FAM-SH-ssDNA, the maximum absorbance peak of AuNPs shifted from 518 nm to 524
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surrounding the modified nanoparticles in Fig. 1(C), which suggests the presence of a coating
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material of lower electron density. The average number of ssDNAs loaded onto each AuNP
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was determined to be 166 ± 9 by the standard curve of fluorescence described above. UV-Vis
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spectroscopy was performed to determine the number of antibodies on the AuNPs. After
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centrifugation, the absorbance of the supernatant was measured at 280 nm and the
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concentration was calculated by the Beer-Lambert Law. The number of antibodies coated on
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the AuNP surfaces was approximately 4.
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3.3 Method establishment
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3.3.1 Optimization of the immune reagents
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The concentrations of the reagents participating in an immunoassay have a strong influence
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on the detection sensitivity and the linear range of competition reactions. A chessboard assay
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was performed to select the working concentrations of the OVA-hapten and AuNP probes.
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The concentrations of OVA-hapten were 0.63, 0.31, 0.16, and 0.08 mg L -1 and the mAb
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concentrations on the AuNP probes were 4.5, 2.2, and 1.1 mg L -1. The results are shown in
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Table 2. In this work, the IC50 (the concentration of analyte that produced 50% inhibition of
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the maximum fluorescence intensity), IC10 (the concentration of analyte that produced 10%
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inhibition of the maximal fluorescence, defined as the limit of detection (LOD)), and ratio of
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maximal fluorescence to IC50 (Fmax/IC50) were used to evaluate the performance of the
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immunoassays. Lower IC50 and IC10 values indicate higher sensitivity, as do higher Fmax/IC50
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values. The optimal concentrations of OVA-hapten and AuNP mAbs were 0.16 and 2.23 mg L
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3.3.2 Optimization of methanol concentration
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Methanol concentrations influence the assay sensitivity by changing the characteristics of the
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analyte solution or affecting the interactions between the mAbs and the hapten coating in the
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well [38]. The methanol concentration was optimized by preparing standard curves of various
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amounts of methanol in 0.01 M PBS. The optimal concentration was selected by comparing
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the IC50, IC10, and Fmax/IC50 of each standard curve. As shown in Fig. 3, the sensitivity
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initially improved with increased methanol concentration, reached a maximum at 10%, then
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decreased thereafter. Thus, 10% methanol-PBS was used as the diluted solution for standards
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and sample extraction.
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FRET between AuNPs (donor) and the 6-FAM fluorophore (acceptor) results in fluorescence
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quenching. Previously published report has shown that DTT displaces thiol-capped
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oligonucleotides from the surface of AuNPs [39]. Herein, DTT was used to displace 6-FAM
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from the AuNPs by ligand exchange, restoring fluorescent emission. The concentration and
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incubation time for DTT were optimized. From Fig. A.3 we can imply that the use of 5 mM
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DTT for 90 min was optimal for the release of the 6-FAM-SH-ssDNAs from the
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hapten-antibody-probe complex.
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3.3.4 Standard curve construction
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ACCEPTED MANUSCRIPT Under optimized conditions, a standard curve of the fluorescence bio-barcode amplification
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immunoassay was generated using inhibition vs. the logarithm of concentration of triazophos.
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As shown in Fig. 4, the competition curve (y = 24.95 x + 65.18, R2 = 0.9738, n=3) showed a
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linear range of detection from 0.01–20 µg L -1. The average IC50 value was 0.25 µg L -1 and
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the LOD was 6 ng L -1. The sensitivity of the established method was over one order of
314
magnitude lower than an indirect competitive ELISA performed under the same laboratory
315
conditions, which had a linear range of 0.1–30 µg L -1, and IC50 and LOD values of 1.1 and
316
0.08 µg L -1, respectively (Fig. A.4).
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3.3.5 Cross-Reactivity (CR)
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The specificity of modified AuNP probes was evaluated by testing the CR with structural
320
analogues of triazophos. Standard solutions (100 mg L-1) of triazophos, chlorpyrifos,
321
chlorpyrifos-methyl, parathion, malathion, diazinon, and fenitrothion were diluted in 10%
322
methanol-PBS to 2000, 500, 100, 20, 5, 1, 0.2, 0.05, and 0.01 µg L -1 for standard curve
323
generation. The CR values were calculated as follows: CR (%) = (IC50 of triazophos/IC50 of
324
analogue) × 100. The structures and results are shown in Table 3. The IC50 values of all six
325
analogues exceeded 2000 µg L -1, and the CRs were < 0.01%. This suggests that the present
326
immunoassay has excellent specificity for triazophos.
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3.3.6 Accuracy and precision
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The accuracy of the fluorescence bio-barcode amplification immunoassay was determined by
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measuring the recovery from samples spiked with triazophos standards at three
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332
standard deviation (RSD) of 5 replicates. Sample extracts were detected by the bio-barcode
333
method, ELISA, and LC-MS/MS simultaneously and the results are shown in Table 4. The
334
average recovery and RSD of the fluorescence bio-barcode immunoassay were 96.1% and
335
12.3%, ranging from 85.0–110.3% and 9.4–17.4%, respectively. The results indicate that the
336
method satisfies the requirements of pesticide residue analytical methods. The average
337
recovery and RSD of the ELISA were 89.1% and 9.0%, ranging from 84.9–98.4% and 6.4–
338
13.2%, respectively, and the average recovery and RSD of the LC-MS/MS were 95.6% and
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1.1%, ranging from 87.1–107.7% and 0.1–2.1%, respectively. Regression analysis was
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performed to compare the detection concentrations of the developed method and LC-MS/MS.
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As compiled in Table 5, a significant correlation has been demonstrated between the two
342
methods for each sample. The square of the coefficients of determination (R2) ranged from
343
0.9709–0.9841 at a confidence level of 95%. These results indicate that the fluorescence
344
bio-barcode amplification immunoassay is a reliable method for triazophos detection.
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3.4 Comparison between various immunoassays
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Indirect competitive ELISA was performed under the same laboratory conditions and the
348
results were compared with that obtained for the competitive fluorescence bio-barcode
349
immunoassay. In terms of operation, the bio-barcode method simplified the steps after
350
immune combination. The fluorescence signal was detected immediately after shocking with
351
DTT, while the ELISA needs to go through the later step of washing, catalyzing, and
352
termination. No matter of analysis time, assay cost or detection accuracy, this method was
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ACCEPTED MANUSCRIPT almost the same as ELISA. High sensitivity is the prominent advantage of this method over
354
indirect competitive ELISA. Finally, this method can achieve multiplex detection using
355
diverse AuNP probes modified with different antibodies and fluorescent oligonucleotides.
356
A detailed comparison of the characteristics of this method and other methods for triazophos
357
detection is provided in Table 6. The current developed method was applied to a variety of
358
samples, including water, fruits, grains, and leafy vegetables. A linear range of more than four
359
orders of magnitude makes this method suitable for a wider concentration range of pesticide
360
residue detection. Methods based on bio-barcoding have lower LODs compared with
361
chemiluminescent enzyme-, fluorescence polarization-, and bead-array competition-based
362
immunoassays. Previous bio-barcoding methods established in our laboratory based on
363
RT-PCR or HRP catalysis were sensitive; however they were complicated, since two types of
364
probes were required for detection. In addition, the wash and separation were operated in a
365
centrifuge tube, which makes it impractical for high numbers of samples’ detection. The
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established method herein has the advantages of high sensitivity and simple operation,
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making it promising for on-site detection of pesticide residues in agro-products and
368
environmental samples.
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4. Conclusion
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Herein, we developed and validated a simple and sensitive immunoassay for triazophos
372
detection using multi-modified AuNPs and fluorescent amplification. The present method
373
was successfully applied to triazophos detection in water, vegetables, fruits, and grains, with
374
low IC50 and detection limit values and a wide working range. This new strategy is promising
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ACCEPTED MANUSCRIPT for the rapid detection of pesticide residues in the environment and agro-products. The
376
competitive fluorescence bio-barcode immunoassay in this study provides a model for the
377
detection of small molecules and has broad prospects for multi-target detection using various
378
AuNP probes with the corresponding antibodies.
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Conflicts of interest: The authors have declared no conflict of interest
381
Acknowledgements
382
This work was supported by the funding from the National Natural Science Foundation of
383
China
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the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy
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of Agricultural Sciences (grant number Y2017JC13).
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Appendix A. Supplementary data
numbers
31671938,
31201371)
and
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(grant
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387 388
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ACCEPTED MANUSCRIPT Scheme 1 (A) Schematic illustration of the AuNP probes preparation. (B) Schematic illustration of the established competitive fluorescence bio-barcode immunoassay for
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pesticide detection.
Fig. 1 Characterization of AuNP probes. (A) The UV-Vis spectrum of bare AuNPs (black line), AuNPs modified with antibodies (red line), and AuNPs modified with
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antibodies and ssDNA (blue line), (B) TEM image of bare AuNPs, and (C) TEM
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Fig. 2 (A) The effects of spacer length on fluorescence intensity and number of ssDNAs labeled on one AuNP probe, and (B) The fluorescence intensity and number
was 0.625 mg L -1.
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Fig. 3 Optimization of methanol concentration; the black, red, and blue lines represent
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the IC50, IC10, and ratio of maximal fluorescence to IC50 (Fmax/IC50) of the standard curve, respectively.
Fig. 4 The standard curve of triazophos; the linear range was 0.01–20 µg L-1; three replicates were performed.
25
ACCEPTED MANUSCRIPT Table 1 Sequence of oligonucleotides used in the present work.
CAGCAGCAGCAGCAGCAGCAGCAAA TTTTTGCAGCAGCAGCAGCAGCAAA TTTTTTTTTTAGCAGCAGCAGCAAA TTTTTTTTTTTTTTTCAGCAGCAAA TTTTTTTTTTTTTTTTTTTTGCAAA TTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTT TTTTTTTTTT TTTTT
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1 2 3 4 5 6 7 8 9 10
Sequence from 5′ to 3′
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ACCEPTED MANUSCRIPT Table 2. Optimized working concentrations of OVA-hapten and antibody adsorbed on the AuNPs surface.
Hapten (mg L-1)
IC50 (µg L-1)
IC10 (µg L-1)
Fmax/IC50
Antibody (mg L-1)
Antibody (mg L-1)
Antibody (mg L-1)
2.23
1.12
4.46
2.23
1.12
4.46
2.23
1.12
0.63
1.19
0.78
0.57
0.19
0.10
0.06
1215.80
764.62
529.91
0.31
0.67
0.90
0.53
0.08
0.12
0.04
2257.47
482.16
468.40
0.16
0.49
0.26
0.54
0.05
0.01
0.05
2660.98
3719.72
408.28
0.08
0.91
0.67
1.0
0.25
0.24
1544.06
1921.90
439.82
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ACCEPTED MANUSCRIPT Table 3. Cross-reactivities (CR%) between triazophos and structural analogues IC50 (µg L-1)
CR (%)
Triazophos
0.25
100
Chlorpyrifos
>2000
Structure
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>2000
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Parathion
<0.01
>2000
<0.01
>2000
<0.01
>2000
<0.01
>2000
<0.01
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ACCEPTED MANUSCRIPT Table 4 Recoveries and relative standard deviations (RSDs) of the bio-barcode immunoassay, ELISA, and LC-MS/MS
Cabbage
Apple
LC-MS/MS
5.0 10.0 50.0 5.0 10.0 50.0 5.0 10.0 50.0 5.0 10.0 50.0 5.0
91.6/17.4 95.3/15.2 102.6/11.1 88.0/10.5 86.8/13.3 90.2/10.6 99.5/13.4 104.7/12.1 92.2/10.6 103.1/10.8 110.3/15.6 107.2/9.4 85.0/10.8
92.7/7.7 98.4/8.2 91.2/9.6 84.9/8.2 90.0/9.7 87.7/7.8 91.7/12.0 88.6/9.3 87.3/6.8 87.2/12.4 87.1/8.0 86.9/6.4 86.3/13.2
98.2/1.1 96.1/0.4 97.0/0.8 88.0/1.2 91.1/0.6 97.3/1.2 87.1/1.0 89.8/1.0 96.1/1.7 96.5/1.3 100.2/1.3 107.7/1.7 98.2/2.1
10.0
96.9/10.4
87.6/9.1
95.1/0.6
50.0
88.2/12.6
89.5/6.4
94.9/0.1
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ACCEPTED MANUSCRIPT Table 5. Regression lines for the fluorescence bio-barcode amplification immunoassay and LC-MS/MS, performed using origin 9.0 at a confidence level of 95%. R2a
Water Rice Cucumber Cabbage Apple
y = 1.0824 x - 0.7413 y = 0.9233 x + 0.3631 y = 0.9262 x + 1.5636 y = 0.9845 x + 0.8142 y = 0.9320 x + 0.2323
0.9794 0.9758 0.9783 0.9841 0.9709
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Regression equation
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Table 6 Comparison of various immunoassays Pattern Method
Sample Solid carrier
FPIAb
Black 96 well plates
96-well Multiscreen HTS
c
BA-IA
plates
Chemiluminometric/Chemilu
HRP-luminol-H2O2 system
minescent detector
Direct competition,
Fluorescence polarization
Water, cowpea,
fluorescein-labeled antigen
values/Multilabel counter
leek
Fluorescence/Bio-Plex
Cabbage,
suspension array reader
carrot, spinach
Indirect competitive, R-PE-conjugated the second antibody Indirect competitive, bio-barcode
d
BBC-IA
magnetic nanoparticles
modified AuNPs probe, PCR
BBC-CIA
magnetic nanoparticles
modified with HPR-labeled bio-barcode, hybridization
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Indirect competitive, AuNPs probe
CFBBCIA
Black 96 well plates
modified with FAM-labeled bio-barcode
Absorbance/Microplate reader
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Indirect competitive, AuNPs, probe f
LOD (µg L-1)
100.7
19.8
0.04-5
0.063
[40]
5-10 min
103.1
10.1
16.09-512
5.860
[41]
2h
84.5
12.3
0.02-50
0.024
[42]
80 min
89.5
15.5
0.04-10
0.020
[25]
45 min
92.1
14.0
0.015-40
0.014
[26]
2.5 h
96.1
12.3
0.01-20
0.006
75 min
Ref.
carrot, water, soil
CT value/Real-time PCR
amplification e
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White 96 well plates
Linear range (µg L-1)
Recovery (%)
Lettuce, apple,
Direct competition,
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Signal/Instrument
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Scheme
RSD (%)
Analysis time
Fluorescence/ Microplate reader
Apple, cabbage, orange, rice
Apple, cabbage, orange, rice Water, rice, cucumber, apple, cabbage
a
Chemiluminescent enzyme immunoassay. b Fluorescent polarization immunoassay. c Bead-array competitive immunoassay.d Bio-barcode amplifcation immunoassay.
e
Colorimetric immunoassay based on bio-barcod. f Competitive fluorescence bio-barcode immunoassay
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This work
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Highlights •
We report a simple and sensitive immunoassay for small molecule detection The effects of spacer length and ligand length on bio-barcode coverage on AuNPs surface were studied
•
Based on competitive binding and bio-barcoded multi-modified gold
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nanoparticles
The immunoassay was comparable to conventional methods in sensitivity
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S0003-2670(17)31209-6
DOI:
10.1016/j.aca.2017.10.032
Reference:
ACA 235506
To appear in:
Analytica Chimica Acta
Received Date: 21 August 2017 Revised Date:
20 October 2017
Accepted Date: 26 October 2017
Please cite this article as: C. Zhang, P. Du, Z. Jiang, M. Jin, G. Chen, X. Cao, X. Cui, Y. Zhang, R. Li, A.M. Abd El-Aty, J. Wang, A simple and sensitive competitive bio-barcode immunoassay for triazophos based on multi-modified gold nanoparticles and fluorescent signal amplification, Analytica Chimica Acta (2017), doi: 10.1016/j.aca.2017.10.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ms. No.: ACA-17-1940
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A simple and sensitive competitive bio-barcode immunoassay for triazophos based on
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multi-modified gold nanoparticles and fluorescent signal amplification
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Chan Zhanga, Pengfei Dub, Zejun Jianga, Maojun Jina, *, Ge Chena, Xiaolin Caoa, Xueyan
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Cuia, Yudan Zhanga, Ruixing Lia, A. M. Abd El-Atyc, d, Jing Wanga, *.
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a
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Agro-Product Quality and Safety, Chinese Academy of Agricultural Sciences; Key Laboratory
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of Agro-Product Quality and Safety, Ministry of Agriculture, Beijing 100081, P. R. China
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Institute of Quality Standard and Testing Technology for Agro-Products, Key Laboratory of
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b
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Key Laboratory od Agro-Products Processing Technology of Shandong Province; Key
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Laboratory of Novel Food Resources Processing, Ministry of Agriculture, 202 Gongye North
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Road, Jinan 250100, P.R. China
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c
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Giza, Egypt
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d
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Konkuk University, Seoul 143-701, Republic of Korea
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Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences;
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Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211
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Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine,
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* Corresponding authors
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Tel.:
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+86-10-8210-6568; Fax: +86-10-8210-6567. E-mail: [email protected] (J. Wang)
+86-10-8210-6570.
E-mail:
[email protected]
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1
(M.
J.
Jin)
and
Tel.:
ACCEPTED MANUSCRIPT Abstract
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A simple and highly sensitive immunoassay based on a competitive binding and bio-barcode
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amplification was designed for detection of small molecules, triazophos. The gold
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nanoparticles (AuNPs) were modified with monoclonal antibodies and 6-carboxyfluorescein
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labeled single-stranded thiol-oligonucleotides (6-FAM-SH-ssDNAs); the fluorescence of
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6-FAM was quenched by AuNPs. Ovalbumin-linked haptens were coated on the bottom of
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microplate to compete with the triazophos in the sample for binding to the antibodies on the
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AuNP probes. The fluorescence intensity was inversely proportional to analyte concentration.
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Parameters of AuNP probes preparation and immune reaction were optimized. At the optimal
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conditions, the salting process was shortened to 1 h and 166 ± 9 ssDNAs were loaded onto a
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single AuNP. The competitive fluorescence bio-barcode immunoassay was performed on
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water, rice, cucumber, cabbage and apple samples. The linear range of the method was 0.01–
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20 µg L-1and the limit of detection (LOD) was 6 ng L-1. The recovery and relative standard
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deviations (RSDs) ranged from 85.0–110.3% and 9.4–17.4%, respectively. Good correlations
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were
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chromatography-tandem mass spectrometry (LC-MS/MS). In conclusion, it is suggested that
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the competitive fluorescent bio-barcode immunoassay had the potential to be used as a
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sensitive method for detection of a variety of small molecules in various samples.
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Keywords Immunoassay; Fluorescence; Bio-barcode; Gold nanoparticles; Triazophos.
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the
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1. Introduction Triazophos (O, O-diethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) phosphorothioate) is a
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ACCEPTED MANUSCRIPT broad-spectrum, moderately toxic insecticide that has been widely used on a variety of crops
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in recent years as a good alternative to highly toxic organophosphates such as parathion,
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parathion methyl, and methamidophos. However, it has a relatively high stability, and its long
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half-life presents potential risks to human health and the environment [1, 2]. There is
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widespread concern over the presence of triazophos residues in environment as well as foods
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[3, 4]. Traditional analytical methods for determination of triazophos include gas
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chromatography (GC) and liquid or gas chromatography coupled with mass spectrometry
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(MS). While these methods are sensitive and accurate, they require expensive equipments,
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skilled technicians, and lengthy analysis [5-8]. In numerous instances, immunoassays have
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proven to be exceptional tools for pesticide detection, as they are simple, cost-effective, and
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rapid. Among immunoassay types, enzyme linked immunosorbent assay (ELISA) is widely
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popular; however, its sensitivity does not meet testing requirements in some cases. Thence,
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the establishment of detection methods with high sensitivity has stimulated extensive
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research.
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Gold nanoparticles (AuNPs) are widely used in the detection and analysis of chemical and
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biological molecules, owing to their unique optical, catalytic, electrical, and chemical
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properties [9-11]. Huo’s group [12] first measured the specific interactions between protein
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A-immobilized AuNPs and human IgG via detecting the average particle size change using
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dynamic light scattering (DLS). Storhoff et al. [13] detected DNA sequences based on the
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colorimetric scattering of AuNP probes. Hou et al. [14] designed an impedimetric
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immunoassay protocol for carcinoembryonic antigen detection based on enzyme-triggered
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formation of tyramine-enzyme repeats on AuNP. In 2003, the bio-barcode technique,
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molecules, was first developed by the Mirkin group to detect and quantify prostate-specific
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antigen [15]. Subsequently, this technique was applied to DNA detection [16, 17]. The system
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relies on the use of magnetic microparticle probes (MMPs) functionalized with antibodies as
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well as AuNPs encoded with bio-barcodes and antibodies. The targets, which are dissolved in
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test solution, are captured by the two probes to form a sandwich construction. The
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bio-barcodes released from the AuNPs are initially determined by silver amplification or
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polymerase chain reaction (PCR). The combination of molecular biology and labeling
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techniques is a hotspot in the detection technology [18-20]. Researchers have modified the
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detection strategy of bio-barcode immunoassay by enzymes, RCA reaction or other schemes
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based on. Liu et al. [21] detected proteins using enzyme-labeled AuNP probes that were
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coated with antibody, single-stranded DNA (ssDNA), and horseradish peroxidase (HRP). Yan
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et al. [22] developed an amplification strategy based on rolling circle amplification (RCA)
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reactions. During the RCA reaction, biotin labels were incorporated into the RCA-generated
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long ssDNA, which bound to avidin-HRP (Av-HRP) to produce enzymatic catalysis-based
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colorimetric signals. Lv et al. [23] modified AuNPs with antibody and G-quadruplex/hemin.
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The hemin released by Exonuclease I can capture the photogenerated electrons to
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significantly amplify the signal intensity. Zhou et al. [24] exploited plasmid-encoded peptide
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tags as surrogate molecules for the matrix-assisted laser desorption/ionization time-of-flight
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MS identification of target DNA.
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It has to be noted that the sandwich construction was not suitable for pesticides and other
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small-molecule haptens, as the latter possess only one antibody-combining site. To conquer
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sandwich construction. In 2015, our group first developed a competitive bio-barcode
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amplification immunoassay for the detection of pesticide residue using quantitative reverse
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transcription (RT)-PCR [25]. This method had a high sensitivity but was time-consuming,
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labor-intensive, and expensive. Afterward, we switched our strategy to a sensitive and
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relatively simple method, in which the AuNPs were modified by antibodies and HRP-labeled
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ssDNA. After magnetic separation, the substrate was added and the absorbance produced by
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the enzyme-catalyzed colorimetric reaction was measured. [26].
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Herein, to further simplify the technique and improve the sensitivity of the previously
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developed bio-barcode immunoassay, we designed a method for triazophos detection using
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only one type of probe. The AuNPs were modified with mAb and 6-FAM-SH-ssDNAs and
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the fluorescence of 6-FAM was quenched by AuNPs. After the competitive reaction between
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ovalbumin-linked
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hapten-mAb-AuNP probe complexes were formed at the bottom of the microplate, as shown
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in Scheme 1. Dithiothreitol (DTT) was used to replace the 6-FAM-SH-ssDNA on the
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complex and the released fluorescence signal was detected.
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2. Experimental section 2.1 Chemicals and materials
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The standard for triazophos (98%) and structural analogues (>95%), bovine serum albumin
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(BSA), goat anti-mouse IgG, polyethylene glycol (PEG) 20000, DTT, Tris-EDTA (TE) buffer
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solution (pH 7.4), hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O, > 99.9%), and
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ACCEPTED MANUSCRIPT trisodium citrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). The triazophos
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hapten and monoclonal antibodies (mAbs) from mouse ascites were generously gifted by the
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Institute of Pesticide and Environmental Toxicology (IPET), Zhejiang University, China.
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Oligonucleotides (3’-FAM, 5’-SH C6) were generated by Shanghai Sangon Biotechnology Co.
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Ltd (Shanghai, China). The 3, 3’ 5, 5’-Tetramethylbenzidine (TMB) ELISA Substrate was
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purchased from TransGen Biotech Co., Ltd (Beijing, China). Octadecylsilyl (C18, 40–60 µm),
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and primary secondary amine (PSA, 40–60 µm) were obtained from Agela (Tianjin, China).
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LC-grade acetonitrile and methanol were supplied by Fisher Scientific (Pittsburgh, PA, USA).
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All other chemicals, such as anhydrous magnesium sulfate (MgSO4) and sodium acetate
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(NaCl) and organic solvents of analytical grade or higher were purchased from Beijing
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Chemical Industry Group Co., Ltd (Beijing, China). Ninety-six-well plates (transparent and
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black, flat bottom) were obtained from Corning, Inc. (Corning, NY, USA). Ultrapure water (≥
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18.2 MΩ·cm) produced by a Milli-Q water purification system (Millipore, Bedford, MA,
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USA) was used in all e experimental works.
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The buffers used in this study were as follows: salting buffer (consisting of 100 mM
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phosphate buffered saline (PBS) and 1.3 M NaCl); blocking and storage buffer (consisting of
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10 mM PBS (pH 7.4), 1% BSA, and 1% PEG 20000); coating buffer (consisting of 50 mM
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carbonate buffer solution (CBS, pH 9.6)); washing buffer (PBST) (consisting of 10 mM PBS
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(pH 7.4) and 0.05% Tween 20); blocking buffer (consisting of 10 mM PBS (pH 7.4) and 2%
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BSA); assay buffer (consisting of 10 mM PBS (pH 7.4) and 0.05% BSA); and ligand
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exchange buffer (comprising 10 mM PBS (pH 8.0)).
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2.2 AuNPs synthesis and modification The 13 nm AuNPs were prepared as previously described [27]. Briefly, 100 mL of 1 mM
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HAuCL4 aqueous solution was heated to boiling with stirring and refluxing, followed by the
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addition of 50 mL of 38.8 mM trisodium citrate rapidly to the boiling solution. The color
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changed from pale yellow to deep red within 5 min; the solution was boiled and stirred for an
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additional 15 min. After the solution was cooled to ambient temperature (25°C), it was
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filtered through a 0.22-µm cellulose nitrate filter. The prepared colloidal particles were
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characterized by UV-Vis spectroscopy (Infinite M200 PRO microplate reader, TECAN,
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Switzerland) and transmission electron microscopy (TEM, TEI Technai G2 F20, Thermo
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Fisher Scientific, MA, USA).
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AuNP probes modified with mAbs and 6-FAM-SH-ssDNA were prepared following a
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previously reported protocol, with minor modifications [16]. The pH value of the AuNP
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solution (1 mL) was adjusted to 9.0 with 30 µL 0.1 M K2CO3, and 18.12 µg mAb was
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thoroughly mixed into the colloidal solution. After withstanding for 1 h, 2 nmol of freshly
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reduced thiolated ssDNA was mixed into the AuNP-antibody complex solution. Subsequently,
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30% PEG 20000 aqueous solution and salting buffer were added to final concentrations of 1%
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and 0.137 M, respectively, followed by incubation at room temperature for 2 h. Thereafter,
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BSA was added to a final concentration of 1% to block the bare surfaces, and the mixture was
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incubated for another 1 h. Finally, the unbound antibody and oligonucleotides were removed
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by two centrifugation steps at 15000 rpm for 20 min at 4°C (Thermo Fisher Scientific, MA,
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USA). The red precipitate was suspended in 200 µL of blocking and storage buffer and could
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stay stable for at least two weeks.
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2.3 Competitive fluorescence bio-barcode amplification immunoassay First, 100 µL per well of OVA-triazophos hapten in CBS was added to a black polystyrene
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microplate and incubated overnight at 4°C. The coated plate was washed three times with
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PBST followed by blocking with 300 µL blocking buffer for 1 h at 37°C. Then 50 µL of
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sample extract or triazophos standard solution, in 10% (v/v) methanol-PBS (0.01 M, pH 7.4),
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and 50 µL AuNP probes, diluted in assay buffer, were sequentially added to the microplate.
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During incubation at 37°C for 1 h, the hapten coating the bottom of the microplate and the
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triazophos in the sample compete for binding with the mAb adsorbed on the surface of the
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AuNPs. When bound, the fluorescence of 6-FAM is quenched by the AuNPs due to
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fluorescence resonance energy transfer (FRET) [28-31]. The unbound pesticide and AuNP
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probes were removed by three washes with PBST. Finally, 100 µL of DTT was added to
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displace the 6-FAM-SH-ssDNA by ligand exchange [16]. The inhibition of fluorescence
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intensity (Ex489nm/Em521nm, Infinite M200 PRO microplate reader, TECAN, Switzerland) and
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the logarithm of the pesticide concentration exhibited a linear relationship.
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2.4 Indirect competitive ELISA
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The coating, blocking, and washing of 96-well transparent polystyrene microplates were
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performed as described above.
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standard solution in 10% (v/v) methanol-PBS (0.01 M, pH 7.4) and 50 µL of diluted mAb
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were sequentially added to the microplate. After incubation at 37°C for 1 h and three washes
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with PBST, 100 µL goat anti-mouse IgG was added, and the plate was incubated at 37°C for
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1 h. Afterward, 100 µL TME was added and the plate was incubated for 15 min at 37°C.
Subsequently, 50 µL of sample extract or triazophos
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Finally, 50 µL of 2 M H2SO4 was added to terminate the reaction and the absorbance at 450
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nm was measured.
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2.5 Sample treatment The samples were tap water from the laboratory, and rice, cucumbers, cabbages, and apples
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bought from a local market in Beijing. All samples were proved to be free from triazophos
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using LC-MS/MS. The water samples were diluted directly with 0.01 M PBS and 10%
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methanol. The pretreatments of other samples were carried out according to the quick, easy,
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cheap, effective, rugged, and safe “QuEChERS” with slight modifications [32]. Briefly, 10 g
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(5 g for rice) samples were weighed into 50 mL centrifuge tubes and mixed thoroughly by
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vortexing for 1 min with 10 mL acetonitrile (5 mL of water was added to the rice samples
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before acetonitrile). After adding 4 g MgSO4 and 1 g NaCl, the mixture was vigorously
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shaken for 1 min and then centrifuged for 5 min at 5000 rpm. Afterward, 2 mL of supernatant
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was transferred to a 10 mL centrifuge tube containing 100 mg PSA, 100 mg C18 and 150 mg
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MgSO4. After vortexing for 1 min, the tubes were centrifuged for 5 min at 10000 rpm. Half of
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the supernatant was transferred to injection vials for LC-MS/MS detection (See Suppl.
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materials) and the other was concentrated under a stream of nitrogen. Finally, the residue was
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dissolved in 10% methanol-PBS and examined by competitive bio-barcode fluorescent
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amplification immunoassay and ELISA.
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3. Results and discussion
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3.1 AuNP probe preparation and optimization
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A solution of 13 nm AuNPs, which exhibited a maximum absorption at 518 nm (Fig. 1(A),
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ACCEPTED MANUSCRIPT black line) was prepared according to the citrate reduction method. The TEM image in Fig.
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1(B) demonstrates that the prepared AuNPs exhibited consistent diameter and did not
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aggregate. The concentration of prepared AuNPs calculated by the Beer-Lambert Law was
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9.9 nM, using an extinction coefficient of 2.47×108 M−1·cm−1 [33]. The gold nanoparticles
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were simultaneously functionalized with 6-FAM-SH-ssDNA and triazophos mAbs. A salt
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aging process is necessary to load the negatively charged DNA onto the citrate ion-adsorbed
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surface of AuNPs. To prevent the irreversible aggregation of colloidal gold, the stepwise
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addition of NaCl often takes one to two days. A previous study indicated that high molecular
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weight PEG stabilizes the system by depletion stabilization, and over 700 DNAs may be
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loaded onto each 50 nm AuNP with 600 mM NaCl within 2 h [34]. Therefore, this experiment
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tested the stability of antibody-modified AuNPs in PEG 20000 by NaCl titration [16]. As
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shown in Fig. A.1 (A), the maximum absorption wavelength displayed a redshift in PEG
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20000 concentrations below 1%, but remained at 524 nm in higher concentrations.
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Comparative tests with the traditional, time-consuming method revealed that PEG 20000
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reduced the irreversible aggregation and the occurrence of oil membrane (Fig. A. 1 (B)). The
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presence of PEG 20000 made the time-consuming salting process unnecessary. The
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6-FAM-SH-ssDNA was maximally loaded after 1 h. It has to be noted that each
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centrifugation step may cause a loss of approximately 10% of AuNPs, and approximately 96%
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supernatant could be removed. Thus, to avoid excessive loss of gold colloidal, two times
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centrifugation are enough to remove most of the unbound materials.
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Previous studies have demonstrated that the nucleobase sequence plays a role in the binding
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of thiol-oligonucleotides to AuNPs, because of the different adsorption affinity of ssDNA
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AuNP surface compared to guanine, cytosine, and alanine deoxyribonucleotides (dG, dC, and
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dA, respectively), which contributes to substantially higher surface coverage of strands with
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(dT)20 than (dA)20 [35,36]. In addition, adsorption of deoxynucleosides, especially dA to gold
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causes the ssDNA strands to lay flat, therefore occupying more surface area than upright
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strands would, and hindering subsequent thiol-ssDNA adsorption [37]. Therefore, to select
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the optimal strand for the probes, various sequences and lengths of ssDNA were used to label
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the AuNP surface, as shown in Table 1. Sequences 1-6 were used to test the sequence effect
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on the coverage and sequences 6-10 were used to optimize the sequence length. The
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fluorescence intensity markedly increased when the spacer increased from (dT)0 to (dT)5, then
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increased slowly as the dT spacer length increased from 5 to 25 bases (Fig. 2(A)). Moreover,
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the fluorescence intensity and ssDNA coverage substantially decreased as ligand length
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increased from 5 to 25 bases (Fig. 2(B)). The number of ssDNA molecules coating each
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particle was calculated by detecting the fluorescence intensity of the supernatant and
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quantifying it based on a standard curve of fluorescence (Fig. A.2) [31]. For a gold solution
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without PEG 20000, long oligonucleotides enhance the surface coverage and stability of
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AuNPs due to steric hindrance [35]. However, interestingly, in the presence of PEG, the
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shorter the oligonucleotides were, the higher the coverage was, since shorter oligonucleotides
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have smaller steric hindrance. Thence, an oligonucleotide sequence of (dT)5 was used to
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prepare the AuNP probes.
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The mAb and thiol-oligonucleotide coverage affects the sensitivity of the method. The
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concentrations of mAb and ssDNA were therefore optimized. Firstly, AuNP probes were
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concentrations of 9.0, 18.1, 36.2, and 72.5 mg L -1 with excess ssDNA (molar rate, 500:1).
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The fluorescence intensity of the supernatant before and after modification was measured and
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the difference represented the amount of ssDNA loaded on the particle surface. Because of
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the increase of surface occupancy by mAbs, the fluorescence intensity decreased gradually
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when the mAbs concentration increased. However, when the probes were added to the
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microplate to combine with the coated triazophos hapten, the maximum fluorescence
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intensity appeared at 36.2 mg L -1. The lower fluorescence value of probes prepared with
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mAb concentrations of 9.0 and 18.1 mg L -1 might be due to the decreased antibodies on the
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particle surface (to form probe-antibody-hapten complexes), despite these probes carrying
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more 6-FAM-SH-ssDNA. Otherwise, the concentration of ssDNAs was optimized by adding
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a series amount of ssDNA to the final molar ratio of ssDNA and AuNPs of 100:1, 200:1,
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300:1, 400:1 and 500:1. As the results shown, the fluorescence increased accordingly as the
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ratio increased from 1:100 to 1:300, and showed a decrease trend as the ratio continued to
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increase to 1:500. Therefore, 18.1 mg L
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6-FAM-SH-ssDNA/AuNPs was used in subsequent experiments.
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3.2 Characterization of AuNP probes
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As shown in Scheme 1, the AuNP probes were modified with triazophos mAbs and
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6-FAM-SH-ssDNA using the optimized conditions and characterized by UV-Vis
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spectroscopy and TEM. Fig. 1(A) shows that after modification with mAbs and
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6-FAM-SH-ssDNA, the maximum absorbance peak of AuNPs shifted from 518 nm to 524
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surrounding the modified nanoparticles in Fig. 1(C), which suggests the presence of a coating
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material of lower electron density. The average number of ssDNAs loaded onto each AuNP
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was determined to be 166 ± 9 by the standard curve of fluorescence described above. UV-Vis
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spectroscopy was performed to determine the number of antibodies on the AuNPs. After
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centrifugation, the absorbance of the supernatant was measured at 280 nm and the
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concentration was calculated by the Beer-Lambert Law. The number of antibodies coated on
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the AuNP surfaces was approximately 4.
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3.3 Method establishment
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3.3.1 Optimization of the immune reagents
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The concentrations of the reagents participating in an immunoassay have a strong influence
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on the detection sensitivity and the linear range of competition reactions. A chessboard assay
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was performed to select the working concentrations of the OVA-hapten and AuNP probes.
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The concentrations of OVA-hapten were 0.63, 0.31, 0.16, and 0.08 mg L -1 and the mAb
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concentrations on the AuNP probes were 4.5, 2.2, and 1.1 mg L -1. The results are shown in
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Table 2. In this work, the IC50 (the concentration of analyte that produced 50% inhibition of
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the maximum fluorescence intensity), IC10 (the concentration of analyte that produced 10%
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inhibition of the maximal fluorescence, defined as the limit of detection (LOD)), and ratio of
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maximal fluorescence to IC50 (Fmax/IC50) were used to evaluate the performance of the
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immunoassays. Lower IC50 and IC10 values indicate higher sensitivity, as do higher Fmax/IC50
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values. The optimal concentrations of OVA-hapten and AuNP mAbs were 0.16 and 2.23 mg L
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3.3.2 Optimization of methanol concentration
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Methanol concentrations influence the assay sensitivity by changing the characteristics of the
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analyte solution or affecting the interactions between the mAbs and the hapten coating in the
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well [38]. The methanol concentration was optimized by preparing standard curves of various
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amounts of methanol in 0.01 M PBS. The optimal concentration was selected by comparing
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the IC50, IC10, and Fmax/IC50 of each standard curve. As shown in Fig. 3, the sensitivity
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initially improved with increased methanol concentration, reached a maximum at 10%, then
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decreased thereafter. Thus, 10% methanol-PBS was used as the diluted solution for standards
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and sample extraction.
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3.3.3 Optimization of DTT
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FRET between AuNPs (donor) and the 6-FAM fluorophore (acceptor) results in fluorescence
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quenching. Previously published report has shown that DTT displaces thiol-capped
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oligonucleotides from the surface of AuNPs [39]. Herein, DTT was used to displace 6-FAM
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from the AuNPs by ligand exchange, restoring fluorescent emission. The concentration and
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incubation time for DTT were optimized. From Fig. A.3 we can imply that the use of 5 mM
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DTT for 90 min was optimal for the release of the 6-FAM-SH-ssDNAs from the
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hapten-antibody-probe complex.
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3.3.4 Standard curve construction
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ACCEPTED MANUSCRIPT Under optimized conditions, a standard curve of the fluorescence bio-barcode amplification
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immunoassay was generated using inhibition vs. the logarithm of concentration of triazophos.
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As shown in Fig. 4, the competition curve (y = 24.95 x + 65.18, R2 = 0.9738, n=3) showed a
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linear range of detection from 0.01–20 µg L -1. The average IC50 value was 0.25 µg L -1 and
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the LOD was 6 ng L -1. The sensitivity of the established method was over one order of
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magnitude lower than an indirect competitive ELISA performed under the same laboratory
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conditions, which had a linear range of 0.1–30 µg L -1, and IC50 and LOD values of 1.1 and
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0.08 µg L -1, respectively (Fig. A.4).
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3.3.5 Cross-Reactivity (CR)
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The specificity of modified AuNP probes was evaluated by testing the CR with structural
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analogues of triazophos. Standard solutions (100 mg L-1) of triazophos, chlorpyrifos,
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chlorpyrifos-methyl, parathion, malathion, diazinon, and fenitrothion were diluted in 10%
322
methanol-PBS to 2000, 500, 100, 20, 5, 1, 0.2, 0.05, and 0.01 µg L -1 for standard curve
323
generation. The CR values were calculated as follows: CR (%) = (IC50 of triazophos/IC50 of
324
analogue) × 100. The structures and results are shown in Table 3. The IC50 values of all six
325
analogues exceeded 2000 µg L -1, and the CRs were < 0.01%. This suggests that the present
326
immunoassay has excellent specificity for triazophos.
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3.3.6 Accuracy and precision
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The accuracy of the fluorescence bio-barcode amplification immunoassay was determined by
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measuring the recovery from samples spiked with triazophos standards at three
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332
standard deviation (RSD) of 5 replicates. Sample extracts were detected by the bio-barcode
333
method, ELISA, and LC-MS/MS simultaneously and the results are shown in Table 4. The
334
average recovery and RSD of the fluorescence bio-barcode immunoassay were 96.1% and
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12.3%, ranging from 85.0–110.3% and 9.4–17.4%, respectively. The results indicate that the
336
method satisfies the requirements of pesticide residue analytical methods. The average
337
recovery and RSD of the ELISA were 89.1% and 9.0%, ranging from 84.9–98.4% and 6.4–
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13.2%, respectively, and the average recovery and RSD of the LC-MS/MS were 95.6% and
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1.1%, ranging from 87.1–107.7% and 0.1–2.1%, respectively. Regression analysis was
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performed to compare the detection concentrations of the developed method and LC-MS/MS.
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As compiled in Table 5, a significant correlation has been demonstrated between the two
342
methods for each sample. The square of the coefficients of determination (R2) ranged from
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0.9709–0.9841 at a confidence level of 95%. These results indicate that the fluorescence
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bio-barcode amplification immunoassay is a reliable method for triazophos detection.
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3.4 Comparison between various immunoassays
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Indirect competitive ELISA was performed under the same laboratory conditions and the
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results were compared with that obtained for the competitive fluorescence bio-barcode
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immunoassay. In terms of operation, the bio-barcode method simplified the steps after
350
immune combination. The fluorescence signal was detected immediately after shocking with
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DTT, while the ELISA needs to go through the later step of washing, catalyzing, and
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termination. No matter of analysis time, assay cost or detection accuracy, this method was
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indirect competitive ELISA. Finally, this method can achieve multiplex detection using
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diverse AuNP probes modified with different antibodies and fluorescent oligonucleotides.
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A detailed comparison of the characteristics of this method and other methods for triazophos
357
detection is provided in Table 6. The current developed method was applied to a variety of
358
samples, including water, fruits, grains, and leafy vegetables. A linear range of more than four
359
orders of magnitude makes this method suitable for a wider concentration range of pesticide
360
residue detection. Methods based on bio-barcoding have lower LODs compared with
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chemiluminescent enzyme-, fluorescence polarization-, and bead-array competition-based
362
immunoassays. Previous bio-barcoding methods established in our laboratory based on
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RT-PCR or HRP catalysis were sensitive; however they were complicated, since two types of
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probes were required for detection. In addition, the wash and separation were operated in a
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centrifuge tube, which makes it impractical for high numbers of samples’ detection. The
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established method herein has the advantages of high sensitivity and simple operation,
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making it promising for on-site detection of pesticide residues in agro-products and
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environmental samples.
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4. Conclusion
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Herein, we developed and validated a simple and sensitive immunoassay for triazophos
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detection using multi-modified AuNPs and fluorescent amplification. The present method
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was successfully applied to triazophos detection in water, vegetables, fruits, and grains, with
374
low IC50 and detection limit values and a wide working range. This new strategy is promising
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ACCEPTED MANUSCRIPT for the rapid detection of pesticide residues in the environment and agro-products. The
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competitive fluorescence bio-barcode immunoassay in this study provides a model for the
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detection of small molecules and has broad prospects for multi-target detection using various
378
AuNP probes with the corresponding antibodies.
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Conflicts of interest: The authors have declared no conflict of interest
381
Acknowledgements
382
This work was supported by the funding from the National Natural Science Foundation of
383
China
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the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy
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of Agricultural Sciences (grant number Y2017JC13).
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Appendix A. Supplementary data
numbers
31671938,
31201371)
and
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387 388
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ACCEPTED MANUSCRIPT Scheme 1 (A) Schematic illustration of the AuNP probes preparation. (B) Schematic illustration of the established competitive fluorescence bio-barcode immunoassay for
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pesticide detection.
Fig. 1 Characterization of AuNP probes. (A) The UV-Vis spectrum of bare AuNPs (black line), AuNPs modified with antibodies (red line), and AuNPs modified with
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antibodies and ssDNA (blue line), (B) TEM image of bare AuNPs, and (C) TEM
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Fig. 2 (A) The effects of spacer length on fluorescence intensity and number of ssDNAs labeled on one AuNP probe, and (B) The fluorescence intensity and number
was 0.625 mg L -1.
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Fig. 3 Optimization of methanol concentration; the black, red, and blue lines represent
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the IC50, IC10, and ratio of maximal fluorescence to IC50 (Fmax/IC50) of the standard curve, respectively.
Fig. 4 The standard curve of triazophos; the linear range was 0.01–20 µg L-1; three replicates were performed.
25
ACCEPTED MANUSCRIPT Table 1 Sequence of oligonucleotides used in the present work.
CAGCAGCAGCAGCAGCAGCAGCAAA TTTTTGCAGCAGCAGCAGCAGCAAA TTTTTTTTTTAGCAGCAGCAGCAAA TTTTTTTTTTTTTTTCAGCAGCAAA TTTTTTTTTTTTTTTTTTTTGCAAA TTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTT TTTTTTTTTT TTTTT
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1 2 3 4 5 6 7 8 9 10
Sequence from 5′ to 3′
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ACCEPTED MANUSCRIPT Table 2. Optimized working concentrations of OVA-hapten and antibody adsorbed on the AuNPs surface.
Hapten (mg L-1)
IC50 (µg L-1)
IC10 (µg L-1)
Fmax/IC50
Antibody (mg L-1)
Antibody (mg L-1)
Antibody (mg L-1)
2.23
1.12
4.46
2.23
1.12
4.46
2.23
1.12
0.63
1.19
0.78
0.57
0.19
0.10
0.06
1215.80
764.62
529.91
0.31
0.67
0.90
0.53
0.08
0.12
0.04
2257.47
482.16
468.40
0.16
0.49
0.26
0.54
0.05
0.01
0.05
2660.98
3719.72
408.28
0.08
0.91
0.67
1.0
0.25
0.24
1544.06
1921.90
439.82
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ACCEPTED MANUSCRIPT Table 3. Cross-reactivities (CR%) between triazophos and structural analogues IC50 (µg L-1)
CR (%)
Triazophos
0.25
100
Chlorpyrifos
>2000
Structure
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>2000
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Parathion
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>2000
<0.01
>2000
<0.01
>2000
<0.01
>2000
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ACCEPTED MANUSCRIPT Table 4 Recoveries and relative standard deviations (RSDs) of the bio-barcode immunoassay, ELISA, and LC-MS/MS
Cabbage
Apple
LC-MS/MS
5.0 10.0 50.0 5.0 10.0 50.0 5.0 10.0 50.0 5.0 10.0 50.0 5.0
91.6/17.4 95.3/15.2 102.6/11.1 88.0/10.5 86.8/13.3 90.2/10.6 99.5/13.4 104.7/12.1 92.2/10.6 103.1/10.8 110.3/15.6 107.2/9.4 85.0/10.8
92.7/7.7 98.4/8.2 91.2/9.6 84.9/8.2 90.0/9.7 87.7/7.8 91.7/12.0 88.6/9.3 87.3/6.8 87.2/12.4 87.1/8.0 86.9/6.4 86.3/13.2
98.2/1.1 96.1/0.4 97.0/0.8 88.0/1.2 91.1/0.6 97.3/1.2 87.1/1.0 89.8/1.0 96.1/1.7 96.5/1.3 100.2/1.3 107.7/1.7 98.2/2.1
10.0
96.9/10.4
87.6/9.1
95.1/0.6
50.0
88.2/12.6
89.5/6.4
94.9/0.1
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ACCEPTED MANUSCRIPT Table 5. Regression lines for the fluorescence bio-barcode amplification immunoassay and LC-MS/MS, performed using origin 9.0 at a confidence level of 95%. R2a
Water Rice Cucumber Cabbage Apple
y = 1.0824 x - 0.7413 y = 0.9233 x + 0.3631 y = 0.9262 x + 1.5636 y = 0.9845 x + 0.8142 y = 0.9320 x + 0.2323
0.9794 0.9758 0.9783 0.9841 0.9709
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Table 6 Comparison of various immunoassays Pattern Method
Sample Solid carrier
FPIAb
Black 96 well plates
96-well Multiscreen HTS
c
BA-IA
plates
Chemiluminometric/Chemilu
HRP-luminol-H2O2 system
minescent detector
Direct competition,
Fluorescence polarization
Water, cowpea,
fluorescein-labeled antigen
values/Multilabel counter
leek
Fluorescence/Bio-Plex
Cabbage,
suspension array reader
carrot, spinach
Indirect competitive, R-PE-conjugated the second antibody Indirect competitive, bio-barcode
d
BBC-IA
magnetic nanoparticles
modified AuNPs probe, PCR
BBC-CIA
magnetic nanoparticles
modified with HPR-labeled bio-barcode, hybridization
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Indirect competitive, AuNPs probe
CFBBCIA
Black 96 well plates
modified with FAM-labeled bio-barcode
Absorbance/Microplate reader
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Indirect competitive, AuNPs, probe f
LOD (µg L-1)
100.7
19.8
0.04-5
0.063
[40]
5-10 min
103.1
10.1
16.09-512
5.860
[41]
2h
84.5
12.3
0.02-50
0.024
[42]
80 min
89.5
15.5
0.04-10
0.020
[25]
45 min
92.1
14.0
0.015-40
0.014
[26]
2.5 h
96.1
12.3
0.01-20
0.006
75 min
Ref.
carrot, water, soil
CT value/Real-time PCR
amplification e
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White 96 well plates
Linear range (µg L-1)
Recovery (%)
Lettuce, apple,
Direct competition,
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Signal/Instrument
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Scheme
RSD (%)
Analysis time
Fluorescence/ Microplate reader
Apple, cabbage, orange, rice
Apple, cabbage, orange, rice Water, rice, cucumber, apple, cabbage
a
Chemiluminescent enzyme immunoassay. b Fluorescent polarization immunoassay. c Bead-array competitive immunoassay.d Bio-barcode amplifcation immunoassay.
e
Colorimetric immunoassay based on bio-barcod. f Competitive fluorescence bio-barcode immunoassay
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Highlights •
We report a simple and sensitive immunoassay for small molecule detection The effects of spacer length and ligand length on bio-barcode coverage on AuNPs surface were studied
•
Based on competitive binding and bio-barcoded multi-modified gold
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nanoparticles
The immunoassay was comparable to conventional methods in sensitivity
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•