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Time-resolved fluorescence immunochromatographic assay developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin and zearalenone in maize and its products Xiaoqian Tang, Peiwu Li, Qi Zhang, Zhaowei Zhang, Wen Zhang, and Jun Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02794 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017
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Analytical Chemistry
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Time-resolved fluorescence immunochromatographic assay developed using two
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idiotypic nanobodies for rapid, quantitative, and simultaneous detection of
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aflatoxin and zearalenone in maize and its products
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Xiaoqian Tang a,b,c,d,e , Peiwu Li a,b,c,d,e*, Qi Zhangd *, Zhaowei Zhang c *,
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Wen Zhang a,b,c , Jun Jiang a
7 8 9 10 11 12 13 14 15 16 17 18 19
a
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences,
Wuhan 430062, China b
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of
Agriculture, Wuhan 430062, China c
Laboratory of Quality & Safety Risk Assessment for Oilseed Products (Wuhan),
Ministry of Agriculture, Wuhan 430062, China d
Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan
430062, China e
Quality Inspection & Test Center for Oilseed Products, Ministry of Agriculture,
Wuhan 430062, China * Corresponding authors at: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, P.R.China
20
Tel.: +86 27 86812943
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Fax:
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E-mail:
[email protected],
[email protected],
[email protected] +86 27 86812862
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ABSTRACT
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Aflatoxins and zearalenone (ZEN) are highly common mycotoxins in maize and
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maize-based products. This study aimed to report a time-resolved fluorescence
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immunochromatographic assay (TRFICA) developed using two idiotypic nanobodies
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for rapid, quantitative, and simultaneous detection of aflatoxin B1 (AFB1) and ZEN in
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maize and its products. A novel Eu/Tb(III) nanosphere with enhanced fluorescence
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was prepared as a label and conjugated to anti-idiotypic nanobody (AIdnb) and
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monoclonal antibody (mAb). On the basis of nanosphere–antibody conjugation, two
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patterns of competitive time-resolved strip methods (AIdnb–TRFICA and mAb–
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TRFICA) were established and compared. The half inhibition concentration of
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AIdnb–TRFICA was 0.46 and 0.86 ng ⋅ mL−1 for AFB1 and ZEN, which was 18.3-
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and 20.3-fold more sensitive than that of mAb–TRFICA for AFB1 and ZEN,
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respectively. Under optimal conditions, AIdnb–TRFICA for dual mycotoxin was
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established and provided a quantitative relationship ranging from 0.13 to 4.54 ng ⋅
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mL−1 for AFB1 and 0.20 to 2.77 ng ⋅ mL−1 for ZEN, with a detection limit of 0.05 and
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0.07 ng ⋅ mL−1 in the buffer solution, respectively. AIdnb–TRFICA showed good
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recoveries (72.6%–106.6%) in samples and was applied to detect dual mycotoxin in
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maize samples with satisfied results. To the best of our knowledge, it is the first report
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about time-resolved strip method based on AIdnbs for dual mycotoxin.
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Analytical Chemistry
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Maize accounts for 30% of global grain production. Its high-yield potential and
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high content of nutrients make it of significant value in human nutrition. However, it
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is prone to be attacked by various molds, including species of Aspergillus and
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Fusarium. Aspergillus and Fusarium are well adapted to maize conditions, making it
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difficult to prevent their growth and possible mycotoxin production in maize.
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Mycotoxins are considered as the most significant chronic dietary risk factor. They
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are secondary metabolites produced by filamentous fungi growing on agricultural
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food during production, processing, and storage.1 Aflatoxins and zearalenone (ZEN)
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are highly common mycotoxins in maize and maize-based products, and may
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co-occur and cause synergistic or additive health effects on the host.2
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Aflatoxins, especially aflatoxin B1 (AFB1), are the most predominant and toxic
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mycotoxins, which are listed as a group I carcinogen by the International Agency for
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Research on Cancer due to their carcinogenic, mutagenic, and teratogenic potential.3
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ZEN is one of the most important Fusarium mycotoxins due to its widespread
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occurrence and toxic properties. One major way to prevent damage from AFB1 and
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ZEN in humans is to detect contaminated food and feed and eliminate them from
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maize and other agricultural products. Thus, developing an approach for determining
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AFB1 and ZEN simultaneously is important.
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Immunoassays are powerful bioanalytical techniques used extensively to
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evaluate food safety for decades. Normally, the competitive immunoassay format is
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used to detect small molecules such as mycotoxins.4, 5The quality of antigens and
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antibodies is essential to the sensitivity, or limit of detection (LOD), which is a crucial
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consideration in immunoassays. The antigen must fulfill a number of requirements
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before being used in mycotoxin assays. First, the mycotoxin must be conjugated to a
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carrier protein, such as bovine serum albumin (BSA), keyhole limpet hemocyanin, 3
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and ovalbumin; also, the coupling rating should be optimized. Second, some of the
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mycotoxins need to be modified as their chemical structure lack the active group
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coupled with the carrier protein directly. Moreover, the modified antigen needs to
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exhibit the specific binding site that can couple with the antibody as much as possible.
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However, the antigen of mycotoxin is synthesized using the mycotoxin standards and
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organic solvent. The mycotoxin standard is expensive and poses a threat to human
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health and environment. Also, the antigen synthesis using the chemical method often
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involves cross-reaction with a similar chemical structure. Therefore, developing a
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specific, environmental-friendly immunoassay that replaces toxic traditional antigen
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is desirable to overcome this limitation.
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Two strategies have been approved as feasible in previous studies. One strategy
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is via the phage display peptide technology.6 It was used to generate mimotopes for
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mycotoxins, such as AFB1,7,8 ZEN,9 DON,10 and OTA.11,12 The peptide mimotope can
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mimic the antibody-binding site instead of mycotoxin conjugates. Nevertheless, it is
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associated with difficulty in controlling the scaffold structure, especially in selecting
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peptides against small proteins. Another strategy is via the development of
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anti-idiotypic antibodies as surrogate antigens. Anti-idiotypic antibodies were
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generated according to primary antibodies, giving rise to humoral immune responses
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in syngeneic or xenogeneic systems.13 The second antibody targets the antigenic
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determinants of the primary antibody. Anti-idiotypic antibodies from camelids gained
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considerable attention due to their unique structure composed of only heavy chains,
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termed as anti-idiotypic nanobodies (AIdnbs).14,15 As early as 1993, they were known
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as a heavy-chain antibody having antigen-binding sites with only three CDRs of the
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variable domain.16 AIdnbs have several advantages, making them extremely suitable
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for use in immunoassays: (1) They have a smaller size, and hence are suitable for 4
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engineering. (2) The type of interaction is shifted from a majority of side-chain
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contacts to main-chain contacts, making them ideal for molecular mimicry.17 (3)
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Compared with traditional anti-idiotypic antibodies, AIdnbs are more likely to
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overcome the matrix interference due to their high solubility and high chemical
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stability. (4) They can be produced on a large scale. The anti-idiotypic antibodies from
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camelids could be an invaluable asset in the engineering of new molecules for
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diagnostic, therapeutic, and biochemical purposes. This technology has also been used
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to generate AIdnbs, which are applied to the detection of mycotoxins. For example,
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Liu et al.18 used OTA AIdnb–alkaline phosphatase fusion proteins and developed a
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fluorescent enzyme immunoassay. The half inhibition concentration (IC50) value of
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the method was 0.13 ng ⋅ mL−1, and the LOD was 0.04 ng ⋅ mL−1. A one-step enzyme
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immunoassay for fumonisin B1 was established using AIdnb–alkaline phosphatase
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fusion proteins; the IC50 and LOD were 2.69 and 0.35 ng ⋅ mL−1, respectively.19
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They also developed an AIdnb against anti-fumonisin B1. A surface plasmon
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resonance assay was used to detect FB1 using this nanobody; the IC50 and LOD were
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0.95 and 0.15 ng ⋅ mL−1, respectively.20 However, all the immunoassays using the
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AIdnbs could be used only for single-component mycotoxin detection, and almost no
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evidence showed whether AIdnbs could work well on an immunochromatographic
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strip.
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The time-resolved fluorescence immunochromatographic assay (TRFICA) used
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lanthanides, such as Eu(III), Tb(III), Sm(III), and Dy(III), as tracers to label the
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antibody with the antigen–antibody reaction. The lanthanide complexes normally
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have a low fluorescence intensity. Therefore, the lanthanides are usually wrapped in
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polystyrene materials or chelate-loaded silica nanoparticles to improve the sensitivity.
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Although fluorescent nanomaterials are especially attractive due to high sensitivity 5
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and good selectivity, they cannot eliminate the background fluorescence, influencing
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the accuracy of detection results. Time-resolved fluorescence has a longer
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fluorescence lifetime that could eliminate the background interference, thus achieving
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more sensitive and specific assays.21,22 Previous studies reported the development of
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the
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determination of AFM1 in raw milk using TRFICA,24 a reliable and sensitive TRFICA
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for OTA in agro-products,25 a monoclonal antibody (mAb)–europium conjugate–based
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lateral flow TRFICA for quantitative determination of T-2 toxin,26 and TRFICA
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determination of carbofuran residues.27 The previous results demonstrated that the
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TRFICAs were rapid and quantitative approaches for detecting contaminants in
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agro-products. However, almost no reports are available on multiple mycotoxin
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detection using TRFICA.
TRFICA
reader23
and
sample-pretreatment-free-based
high-sensitivity
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Therefore, based on phages 2–5 and 8# AIdnbs, this study explored the
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feasibility of combining AIdnbs and TRFICA to develop a method for detecting AFB1
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and ZEN simultaneously.
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EXPERIMENTAL SECTION
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Chemicals and materials
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AFB1, AFB2, AFG1, AFG2, AFM1, ZEN, β-zearalenol standards,
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1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), boric acid, and BSA were all
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purchased from Sigma–Aldrich (MO, USA). mAb for aflatoxin (1C11)28 and ZEN
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(2D3) , 29TOP10F′ cell-carrying VHH expression plasmid (named phages 2–5 and
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8#),30 rabbit anti-mouse immunoglobulin G (IgG), and goat anti-apical IgG were
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produced in the laboratory. The xTractor buffer for protein extraction and His60
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Superflow Resin were purchased from Clontech Laboratories, Inc. (CA, USA).
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Further, 0.01M phosphate-buffered saline (PBS, pH 7.4) was prepared by adding 8 g 6
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of NaCl, 2.9 g of Na2HPO4⋅12H2O, 0.2 g of KH2PO4, and 0.2 g of KCl in 1000 mL of
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deionized water. All organic solvents and inorganic chemicals were of reagent grade.
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Methanol, formic acid, and acetonitrile of high-performance liquid chromatography
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grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
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Unless otherwise stated, the other inorganic chemicals and organic solvents were of
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analytical reagent grade or better. Ultrapure water was obtained from a Milli-Q water
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purification system (Millipore Co., Ltd., MA, USA). Nitrocellulose membranes,
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sample pads, and absorbent pads were purchased from Millipore Corp. (MA, USA).
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Apparatus
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The Varioskan Flash (Thermo Fisher Scientific, MA, USA), AnXYZ3050
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Dispensing Platform, CM4000 Guillotine Cutter, and LM4000 Batch Laminator
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(BioDot, CA, USA) were used to prepare test strips. The high-speed freezing
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centrifuge (CF16RX) was purchased from Hitachi (Tokyo, Japan). A homemade
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portable fluorescence spectrophotometer was employed. A Xe lamp served as the
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excitation source at 365 nm. The signal acquisition was obtained at 613 nm. A typical
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delay of 400 µs occurred when the emission light was collected from the excited light
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source.
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Purification of the AIdnbs for AFB1 and ZEN
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The TOP10F′ cells carrying the variable domain of the heavy chain (VHH)
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expression plasmids (named phages 2–5 and 8#) were inoculated into the LB
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ampicillin agar plates and cultured at 37°C overnight. The clone growing on the
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semisolid medium was transferred to a 2.5 mL of SB medium and cultured at 37°C
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and 250 rpm. When the SB medium achieved an optical density of 0.6–0.8, it was
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added to 250 mL of the SB medium containing 100 µg/mL of ammonia benzyl. Then,
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250 µL of isopropyl β-D-1-thiogalactopyranoside (1mM) was added, followed by 7
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continuous culture with shaking overnight. The AIdnbs were purified following the
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manufacturer’s instruction. The nanobodies containing 6× His tag were purified with
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Ni-chelating affinity chromatography. The purified AIdnbs were further analyzed by
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sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis according to a
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standard protocol. The concentrations were determined using the Bradford method.
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Preparation and characterization of Eu/Tb(III) nanospheres
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The Eu/Tb(III) nanospheres were prepared according to a reported method
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modified to enhance the fluorescence. First, the carboxyl polystyrene nanospheres
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were prepared. Then, 10 mmol/L of styrene monomer and 0.95 mmol/L of acrylic
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monomer were diluted with 10 mL of deionized water containing 0.45mM SDS in a
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round-bottom flask. After removing the air with high-purity nitrogen, 0.5 mL of
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0.15mM potassium persulfate was added into the flask at 70°C and stirred for 8 h. The
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mixture was filtered and dialyzed for 5 days. Second, for preparing enhanced
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Eu/Tb(III) nanospheres, the carboxyl polystyrene nanospheres were diluted with 10
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mL of acetone solution (acetone:deionized water = 1:1, v/v), and then 100 µL of
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EuCl3 (0.1 mol ⋅ L−1), 100 µL of TbCl3 (0.1 mol ⋅ L−1), 300 µL of trioctylphosphine
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oxide, 100 µL of phenanthroline, and 400 µL of β-NTA (0.1 mol ⋅ L−1) were added.
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The reaction occurred in the dark for 10 h at 60°C and continued for another 2 h at
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room temperature. After removing the organic solvents, the nanosphere solution was
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dialyzed for 5 days. Finally, the obtained nanosphere solution was stored at 4°C for
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further analysis and use.
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Preparation and characterization of Eu/Tb(III) nanosphere probe
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Two kinds of Eu/Tb(III) probes, including AIdnb labeled with nanospheres (AIdnb
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probe) and mAb labeled with nanospheres (mAb probe), were prepared as follows.
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Boric acid buffer solution (400 µL) was mixed with 100 µL of Eu/Tb(III) nanospheres. 8
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After sonicating for 6 s, the EDC solution (15 mg ⋅ mL−1) was added and mixed for 15
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min. The suspension was separated by centrifugation at 13,000 rpm for 10 min. The
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upper aqueous layer was removed, and the residue was resuspended in 0.5 mL of
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boric acid buffer using a sonicator for 6 s. Then, 10, 20, and 40 ng of antibodies,
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including phages 2–5 and 8#, 1C11, and 2D3, were added. The mixture was shaken
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for 2 h before being separated by centrifugation at 13,000 rpm for 10 min. The upper
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aqueous layer was removed and transferred to a new tube for studying the coupling
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ratio of nanospheres and antibodies. The residue was resuspended in 0.5 mL of 0.5%
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BSA/boric acid buffer. After the reaction continued for another 0.5 h, the probe
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solution was stored at 4°C. Four nanosphere probes, including phages 2–5 and 8#,
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1C11, and 2D3, were analyzed by Fourier transform infrared spectroscopy (FTIR),
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transmission electron microscopy (TEM), and energy-dispersive spectrometry (EDS).
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Two patterns of competitive TRFICA
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The test strips comprised a typical absorbent pad, nitrocellulose (NC) membrane,
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and sample pad as previously described;31 the details are given in supplementary
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information. AFB1 and ZEN are small molecules. In the immunoassay, a target
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compound was needed to compete with the antigen for binding to the antibody. Two
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patterns of competitive TRFICA were established. For mAb–TRFICA, 1C11 and 2D3
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solutions were coated on the NC membrane as capture antigens, and phages 2–5 and
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8# probes were used as detectors. The sensitivity and LOD of the two patterns were
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analyzed. For AIdnb–TRFICA, phages 2–5 and 8# were coated on the NC membrane
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as capture antigens, and 1C11 and 2D3 probes were used as detectors. On the T line,
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the target compounds and phage 2–5 or 8# would compete for the binding site of
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1C11 and 2D3 probes.
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Analytical procedure of AIdnb–TRFICA for dual mycotoxin 9
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The dual strip comprised one C line and two T lines on the NC membrane. The T
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lines were coated with phages 2–5 and 8#, and the C line was coated with rabbit IgG.
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AIdnb–TRFICA underwent the following procedure. The sample solution (75 µL) and
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probe solution (75 µL) were diluted with analysis buffer and added into the microwell
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(n = 3). The strip was inserted into the microwell and detected using a homemade
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apparatus after reacting for 8 min at 37°C. Six kinds of analysis buffer, including 1%
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PVPK 30 + 1% sucrose + ddH2O, 1% PVPK 30 + 1% BSA + ddH2O, 1% PVPK 30 +
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2% sucrose + 1% Tween 20 + ddH2O, 2% sucrose + 1% Tween 20 + ddH2O, and 2%
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sucrose + 1% PVPK 30 + ddH2O, were used. Therefore, the amounts of antigen on T
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and C lines and dilution of antibody probes were optimized.
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RESULTS AND DISCUSSION
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Characterization of the Eu/Tb(III) nanospheres and nanosphere probes
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The fluorescence intensities of obtained nanospheres were compared with a method
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used in a previous study.31 The molar concentration was calculated according to the
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following equation:
c=
235
ேఽ
236
where NA is the Avogadro constant, n is the number of nanospheres, and V is the
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volume of nanospheres. The Varioskan Flash was used to measure the value of
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fluorescence intensities. Figure 1a shows that at the same molar concentration, the
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fluorescence intensity of Eu/Tb(III) nanospheres was 18,000, higher than that of the
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nanospheres without Tb(III). This was attributed to the co-luminescence effect of
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Tb(III), indicating that the Eu/Tb(III) nanosphere provided a satisfactory fluorescence
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intensity.
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The optical properties of Eu/Tb(III) nanospheres were identified with
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fluorescence spectra. Figure 1b shows that when the nanospheres were excited at 350 10
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nm, a maximum emission peak appeared at 618 nm. The Eu/Tb(III) nanospheres
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displayed a strong red photoluminescence emission under the ultraviolet lamp.
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The FTIR, TEM, and EDS of Eu/Tb(III) nanospheres before and after coupling
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with antibodies were performed to study the characterization of nanosphere probes.
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The FTIR spectra of the nanospheres and nanosphere probes are illustrated in Figure
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2(a–e). FTIR had a high sensitivity to small variations in hydrogen-bonding patterns,
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making amide I (1600–1700 cm–1) band uniquely useful for the analysis of protein
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secondary structure.32 Curve a was the absorption spectrum of the blank Eu/Tb(III)
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nanospheres. The absorption of stretching vibration at 1602 cm–1 revealed that
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carboxyl functional groups were successfully decorated on the surface of Eu/Tb(III)
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nanospheres. No obvious absorption peak appeared in the blank Eu/Tb(III)
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nanospheres at 1600–1700, 1480–1575, and 1220–1330 cm–1, indicating that the
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blank Eu/Tb(III) nanospheres were not contaminated with proteins. The spectrum of
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Eu/Tb(III) nanospheres (curves b, c, d, and e) conjugated to antibodies showed a clear
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signal within 1600–1700 cm–1 corresponding to the amido bond of antibodies,
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indicating that the antibodies were conjugated to the Eu/Tb(III) nanospheres. Figure
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2f shows the TEM images of the as-prepared Eu/Tb(III) nanospheres conjugated to
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antibodies. The nanospheres were spherical in shape with a size of 190 nm. Figure 2f
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(2–5) illustrates a layer of protein-wrapped nanospheres. Figure 2f (4 and 5) shows
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that a smaller area of protein wrapped the nanospheres compared with that in Figure
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2f (2 and 3) due to the lower molecular weight of AIdnbs. The differences in TEM
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between the nanospheres and nanosphere probes demonstrated that the AIdnb and
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mAb probes were successfully conjugated. The antibodies labeled with nanospheres
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were evidenced by comparing the EDS of nanospheres before and after conjugation,
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as shown in Figures 2g and 2h. Characteristic peaks of C and N were obviously higher 11
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in the spectrum of nanospheres after conjugating with antibodies.
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Optimizing the nanosphere probe
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Gel electrophoresis was used to detect the size of AIdnb; it was about 15 kDa,
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consistent with the theoretical value (Fig. S1). The concentration of AIdnb was
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determined using the Bradford method; 2.3 mg/L of phage 2–5 and 3.5 mg/L of phage
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8# were obtained. The amino acid sequences of phages 2–5 and 8# were analyzed
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using the ProtParam software (http://web.expasy.org/protparam/) in the online
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software tool ExPASy system (http://www.expasy.org/). The isoelectric point of
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phages 8# and 2–5 was 5.81 and 6.70, respectively. Thus, the pH of the buffer
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solution used during the conjugation should be higher than that at least.
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The amounts for each AIdnb and mAb value were adjusted in the production of two
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kinds of probes. Three concentrations, 10, 20, and 40 ng, of AIdnb (phages 2–5 and
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8#) and mAb (1C11 and 2D3), were conjugated to the Eu/Tb(III) nanospheres. The
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coupling rate (CE) was evaluated according to the following formula: CE (%)
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=
285
antibodies, having a CE between 32% and 80.8%. As shown in Figure S2a, the
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fluorescence intensity of the T line could reach 10,000 when the CE was between 32%
287
and 80.8% after appropriate dilution. Therefore, 10 ng of antibody was sufficient in
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conjugation. A view of how the pH of boric acid buffer solution and amounts of EDC
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affected the fluorescence intensity is given in Figure S2b and S2c. For the 1C11, 2D3,
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and phage 8# probes, the fluorescence intensity value on the T line was almost equal
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at pH 8.12–8.5. The phage 2–5 probe had a good fluorescence intensity at pH 8.5–9.0.
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Different amounts, 37.5, 75, 125, and 750 ng, of EDC solution (15 mg ⋅ mL−1) were
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examined in terms of performance to understand their remarkable effect on the
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nanosphere probes. For the phages 2–5 and 8# probes, the low fluorescence intensity
୮୰ୣି୵ୟୱ୦ ୮୰ୣ
× 100. The value of CE increased with the increase in the amounts of
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of T line was obtained from 37.5 and 750 ng, with no significant difference between
296
125 and 750 ng. For the 1C11 and 2D3 probes, the fluorescence intensity of T line
297
increased between 37.5 and 125 ng with the increase in the amounts of EDC. Hence,
298
75 ng of EDC was chosen for phages 2–5 and 8# probe conjugation, and 125 ng of
299
EDC was chosen for 1C11 and 2D3 probe conjugation.
300
Comparison of two patterns of TRFICA for mycotoxin
301
The sensitivities of AIdnb–TRFICA and mAb–TRFICA were compared to establish
302
a TRFICA method for AFB1 and ZEN. The target (AFB1 or ZEN) competed for the
303
binding site of 1C11 and 2D3 probes with AIdnb on the T line in the AIdnb–TRFICA
304
pattern (Scheme S1a). AFB1 and ZEN competed for the binding site of 1C11 and 2D3
305
on the T line with AIdnb probes in the mAb–TRFICA pattern (Scheme S1b). The
306
experimental parameters of the two patterns were optimized for the AFB1 strip as
307
follows: For the mAb–TRFICA pattern: phage 2–5 probe dilution factor was 100×; 1
308
mg ⋅ mL−1 (1C11) × 0.7 µg ⋅ cm−1 for the T line, and 1 mg ⋅ mL−1 (anti-apical IgG) ×
309
0.7 µg ⋅ cm−1 for the C line. For the AIdnb–TRFICA pattern: 1C11 probe dilution
310
factor was 150×; 1 mg ⋅ mL−1 (phage 2–5) × 0.7 µg ⋅ cm−1 for the T line, and 0.1 mg ⋅
311
mL−1 (rabbit anti-mouse IgG) × 0.4 µg ⋅ cm−1 for the C line. The series of a standard
312
solution of AFB1 (60, 30, 20, 10, 5, 1.65, 0.55, 0.165, and 0.055 ng ⋅ mL−1) were
313
prepared, and a standard curve was established between the value of fluorescence
314
intensity and concentration of AFB1. The calculated sensitivity (IC50) of AIdnb–
315
TRFICA and mAb–TRFICA was 0.46 ng ⋅ mL−1 and 8.42 ng ⋅ mL−1, respectively, for
316
AFB1 (Fig. 3a).
317
The experimental parameters of the two patterns for the ZEN strip were optimized
318
as follows. For the mAb–TRFICA pattern: the phage 8# probe dilution factor was
319
200×; 1 mg ⋅ mL−1 (2D3) × 0.7 µg ⋅ cm−1 for the T line, and 1 mg ⋅ mL−1 (anti-apical 13
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320
IgG) × 0.7 µg ⋅ cm−1 for the C line. For the AIdnb–TRFICA pattern: 2D3 probe
321
dilution factor was 300×; 1 mg ⋅ mL−1 (phage 8#) × 0.7 µg ⋅ cm−1 for the T line, and
322
0.1 mg ⋅ mL−1 (rabbit anti-mouse IgG) × 0.4 µg ⋅ cm−1 for the C line. Different
323
concentrations of standard solution of ZEN (80, 26.6, 8.8, 2.96, 0.98, 0.33, 0.10, and
324
0.033 ng ⋅ mL−1) were prepared. Standard curves using the mAb–TRFICA and AIdnb–
325
TRFICA patterns were plotted for detecting ZEN; the IC50 was 17.48 and 0.86 ng ⋅
326
mL−1, respectively (Fig. 3b).
327
These results indicated that AIdnb–TRFICA possessed an 18.3-fold higher
328
sensitivity than that of mAb–TRFICA for AFB1, and a 20.3-fold for ZEN. A possible
329
reason for this was as follows.
330
In the AIdnb–TRFICA format, targets (AFB1 or ZEN) bound to the specific
331
antibody (1C11or 2D3) first and then the competitive reaction occurred on the T line,
332
suggesting that the binding reaction time for the free targets was longer than that for
333
the immobilized antigen (phages 2–5 or 8#) and that the “unfairness” occurred in this
334
format and the specific antibody tended to bind to the free targets from the tested
335
sample. This unfairness resulted in a higher sensitivity. However, it was different in
336
the mAb–TRFICA format. The competitive reaction just occurred on the T line, and
337
both labeled antigens (phage 2–5 or 8#) and the free targets from the tested sample
338
competed fairly at almost the same time on the T line.
339
Therefore, the AIdnb–TRFICA pattern was chosen for further research.
340
AIdnb–TRFICA for dual mycotoxins
341
The competitive TRFICA for AFB1 and ZEN was a dual strip in which the different
342
lines expressed different kinds of mycotoxins. The phages 2–5 and 8# nanobodies
343
were coated on the NC membrane as T1 and T2 lines, respectively, and 1C11 and 2D3
344
probes were used as detectors. The rabbit anti-mouse IgG was coated on the NC 14
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345
membrane as the C line. The principle of AIdnb–TRFICA was analogous to the
346
traditional TRFICA principle found in the previous studies.26 AIdnb–TRFICA did not
347
need to use highly toxic mycotoxin antigens. For negative samples, 1C11 and 2D3
348
probes flowed toward the T1 and T2 lines, where the probes were captured by the
349
phages 2–5 and 8# immobilized on the T lines. The excess probes flowed toward the
350
C line and were captured by the rabbit anti-mouse IgG on the C line. For positive
351
samples, the targets (AFB1 and ZEN) in the samples reacted with 1C11 and 2D3
352
probes; less or none 1C11 and 2D3 probes were captured on the T1 and T2 lines. The
353
higher the concentration of the target compounds in the sample, the less the probes
354
captured on the T line (Scheme 1). Eu/Tb nanospheres had a fluorescence emission at
355
613 nm upon excitation at 365 nm; the fluorescence signals collected from the T and
356
C lines were proportional to the amounts of AFB1 and ZEN. As expected, an increase
357
in the amounts of AFB1 and ZEN in the sample solution resulted in a dynamic
358
decrease in the fluorescence emission intensity of T/C (Fig. S3c). Obvious signals
359
were obtained in the negative sample, whereas the positive ones displayed no signals
360
on T1 and T2 lines (Fig. S3a and S3b).
361
Experimental parameters, including the concentration of immunoassay reagents,
362
analysis buffer, dilution factor of probes, and concentration of methanol in the extract
363
sample solution, were optimized to improve the sensitivity of AIdnb–TRFICA. The
364
highest sensitivity was obtained under the optimal conditions as follows:
365
concentration of immunoassay reagents, 1.0 mg ⋅ mL−1 × 0.75 µg ⋅ cm−1 for T1 and
366
0.5 mg ⋅ mL−1 × 0.6 µg ⋅ cm−1 for T2; 0.05 mg ⋅ mL−1 × 0.4 µg ⋅ cm−1 for C line.
367
Finally, 2% sucrose + 1% Tween 20 + ddH2O was selected as analysis buffer, which
368
could give a clean signal on the T and C lines, with less background interference (Fig.
369
S4). The dilution factor for 1C11 and 2D3 probes was 150× and 300×, respectively. 15
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370
Further, 0%, 5%, 15%, 25%, 45%, and 65% methanol–water (v/v) were used as
371
sample solutions to study the effect of methanol in AIdnb–TRFICA (Fig. S5). The
372
value of T1/C and T2/C slightly decreased with the increase in methanol
373
concentration till 15%. Further increasing the methanol concentration led to a
374
dramatic decline in T/C. Satisfactory results were obtained between 5% and 15%
375
methanol–water (v/v). This result was different from the one previously reported that
376
nanobodies had a good tolerance to organic solvents in enzyme-linked
377
immunosorbent assay (ELISA),33 indicating that nanobodies showed a lower tolerance
378
to organic solvents in AIdnb–TRFICA.
379
A linear relationship between the value of T/C and log (analytical concentration)
380
of AIdnb–TRFICA was calculated.34 The LOD, IC50, and dynamic range are shown
381
in Table 1. The experiments indicated that the proposed technique could be used for
382
simultaneous determination of AFB1 and ZEN.
383
Other mycotoxins, including AFB2, AFG1, AFG2, AFM1, and β-zearalenol, were
384
used in AIdnb–TRFICA to evaluate cross-reactivity (Fig. 3e and 3f). The
385
cross-reactivity with AFB2, AFG1, AFG2, AFM1, and β-zearalenol was 85.7%, 51.8%,
386
31.1%, 19.4%, and 78.1%, respectively, which was consistent with the results in
387
previous studies using ELISA29, 30.
388
Validation of the developed AIdnb–TRFICA for dual mycotoxin
389
The sample preparation was similar to published extraction methods,35 modified
390
with the intention of less danger for the operator and ease of execution for a rapid
391
detection. First, 10, 20, and 50 ng of AFB1 and 20, 100, and 500 ng of ZEN were
392
added to 5.0 g maize sample (ground to a powder, through 20 meshes) in a 50-mL
393
glass tube and allowed to stand at 4°C overnight. After adding 20 mL of methanol–
394
water (80:20, v/v) solution to each sample, the samples were vortex-mixed for 0.5 min 16
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395
and ultrasonicated at 50°C for 10 min. The samples were then centrifuged at 6000
396
rpm for 5 min. The upper clear layer was subsequently passed through a 0.45-µm
397
filter membrane. The extracts were five times diluted with PBS (pH 7.4) before
398
analysis by AIdnb–TRFICA.
399
Matrix calibration curves were established for validation evaluation (Fig. 3c and
400
3d). The recovery study was carried out by calculating the ratio of the concentration
401
value to the spiking concentration.36 Three levels of spiking concentration for AFB1
402
and ZEN were analyzed by AIdnb–TRFICA, as summarized in Table S2. The
403
precision was estimated by the coefficient of variation (CV) analyzed from the results
404
with five parallel determinations. The comparison between the results revealed that
405
the mean recoveries were 72.6%–106.6% for AFB1 and 75.6%–91% for ZEN, with
406
CV ranging from 5.4% to 9.6% and 5.8% to 10.6%, respectively.
407
Application in nature-contaminated samples
408
A total of nine maize and maize flour samples were analyzed using the AIdnb–
409
TRFICA method and liquid chromatography–mass spectrometry (LC–MS/MS). The
410
detail of LC–MS/MS method is provided in supplementary information. The
411
coefficient of determination values obtained was between 0.9701–0.9986 for AFB1
412
and 0.9832–0.9995 for ZEN. As shown in Table 2, the standard deviation and
413
coefficient of variation were acceptable and satisfactory.
414 415 416
Hence,
the
established
AIdnb–TRFICA
method
provided
an
environmental-friendly tool for detecting mycotoxin in maize. CONCLUSIONS
417
Eu/Tb(III) nanosphere synthesis and nanosphere–probe conjugation were clearly
418
demonstrated in this study. The pH, amounts of antibody, and EDC were optimized
419
during the conjugative reaction, rendering nanosphere probes suitable for 17
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420
immunoassay detection for mycotoxin. Two novel patterns of TRFICA method based
421
on mAb and AIdnb for detecting mycotoxins were developed. The AIdnb–TRFICA
422
pattern was finally selected for simultaneous detection, since it exhibited a 10- to
423
20-fold more sensitivity than mAb–TRFICA for AFB1 and ZEN. Notably, the AIdnb–
424
TRFICA provided practical reliability and sensitivity in the quantitative, simultaneous
425
immunoassay without using the highly toxic antigen. Furthermore, AIdnb could be
426
used in the strip method and might have potential applications for other mycotoxins.
427 428 429
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31471651).
430
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X.. Talanta 2015,143, 388-393.
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23. Zhang, Z. W.; Tang X. Q.; Wang D.; Zhang Q.; Li P. W.; Ding X. X.. PloS one
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2015, 10, e0123266.
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24. Tang X. Q.; Zhang, Z.; Li P. W.; Zhang Q.; Jiang J.; Wang D.; Lei J. W.. RSC
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25. Majdinasab, M.; Mahmoud S. Z.; Sabihe S. Z.; Pei W. L.; Zhang Q.; Li X.; Tang
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X. Q.; Li J.. Food Control 2015, 47, 126-134.
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26. Zhang, Z. W.; Wang D.; Li J.; Zhang Q.; Li P. W.. 2015, Analytical Methods 2015,
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27. Zhang, Q.; Qu, Q.; Chen, S.; Liu, X.; Li, P.. Food Chem 2017,231, 295-300.
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28. Zhang, D. H.; Li, P. W.; Zhang, Q.; Zhang, W.; Huang, Y. L.; Ding, X. X.; Jiang,
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29. Tang, X. Q.; Li, X.; Li, P.; Zhang, Q.; Li, R.; Zhang, W.; Ding, X.; Lei, J.; Zhang,
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30. Wang, Y.; Dechant, J.E.; Gee, S.J.; Hammock, B.D.. Anal Chem 2013,85,
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31. Wang, D.; Zhang, Z.; Li, P.; Zhang, Q.; Ding, X.; Zhang, W.. J Agric Food Chem
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32. Kong J.; Yu S.. Acta Biochimica Et Biophysica Sinica 2007, 39: 549-559.
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33. He, T.; Wang, Y.; Li, P.; Zhang, Q.; Lei, J.; Zhang, Z.; Ding, X.; Zhou, H.; Zhang,
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34. Wang, Y. K.; Yan Y. X.; Ji W. H.; Wang, H. A.; Li, S. Q.; Zou, Q.; Sun, J. H.. J
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Agric Food Chem 2013, 61, 5031-5036.
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35. Li, Q.; Lu, Z.; Tan, X.; Xiao, X.; Wang, P.; Wu, L.; Shao, K.; Yin, W.; Han, H..
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501
Table 1. Analytical characteristics for AFB1 and ZEN determination using
502
AIdnb–TRFICA in buffer solution
LOD, ng
IC50, ng
IC20-IC80, ng
mL-1
mL-1
mL-1
AFB1
0.05
0.53
0.13-4.54
y=1.48/(1+x/0.18)^0.58
ZEN
0.07
0.52
0.20-2.77
y=0.06+0.88/(1+x/0.50)^0.92
Equation
503 504
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505
Table 2. Comparison of the results of dual mycotoxin in naturally contaminated
506
samples using AIdnb–TRFICA and LC–MS/MS AIdnb-TRFICA, mean±SDa (ng mL-1)
Sample
LC-MS /MS, mean±SD (ng mL-1)
AFB1
CVb(%)
ZEN
CV (%)
AFB1
CV (%)
ZEN
CV (%)
1
15.6±6.5
8.6
26.6±4.6
9.8
14.5±2.1
8.9
29.1±1.8
9.0
2
10.3±3.4
8.5
32.9±3.7
8.5
9.2±2.4
10.5
34.6±1.2
9.5
3
12.6±3.4
9.5
28.1±3.8
8.9
12.3±3.1
9.5
30.6±2.8
11.5
1
4.6±3.7
11.2
33.3±3.9
7.8
3.8±2.9
13.6
35.6±2.7
11.2
2
1.6±3.4
13.2
46.0±3.5
8.8
1.1±2.6
13.5
48.6±0.9
10.6
3
4.3±3.7
9.6
34.3±2.7
8.1
3.7±1.9
11.0
36.3±1.7
7.2
1
30. 5±5.4
8.2
49.9±5.1
7.9
24.0±0.6
8.1
50.8±4.1
9.9
2
20.6±3.5
7.8
52.6±4.4
8.5
21.1±0.7
8.6
55.8±3.8
7.2
3
9.1±3.1
7.5
35.9±4.0
7.2
7.8±3.3
9.0
38.3±3.5
8.2
Yellow maize
White maize
Maize flour
507
a
Standard deviation.
508
b
Coefficient of variation.
509
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510 511
Figure 1. (a) Comparison of fluorescence intensity of Eu/Tb nanospheres with
512
other nanospheres. (b) Fluorescence excitation (350 nm) and emission spectra
513
(618 nm) of Eu/Tb nanospheres.
514
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515 516
Figure 2. (a) FTIR spectra of the Eu/Tb nanospheres, (b) 1C11 probes, (c) 2D3
517
probes, (d) 2–5 probes, and (e) 8# probes. (f) TEM images of the (1)
518
Eu/Tb-nanospheres, (2) 1C11 probes, (3) 2D3 probes, (4) 2–5 probes, and (5)
519
8# probes. (g) EDS of spectrogram of Eu/Tb nanospheres and (h) nanosphere
520
probes. 25
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521 522 523
Scheme 1. Schematic illustration of AIdnb–TRFICA for dual mycotoxin.
524
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525 526
Figure 3. (a) Comparison of AIdnb–TRFICA and mAb–TRFICA for AFB1. (b)
527
Comparison of AIdnb–TRFICA and mAb–TRFICA for ZEN. (c) Standard curve
528
of AIdnb–TRFICA for AFB1. (d) Standard curve of AIdnb–TRFICA for ZEN. (e)
529
Cross-reaction of AIdnb–TRFICA for AFB1. (f) Cross-reaction of AIdnb–
530
TRFICA for ZEN.
531 532 533 27
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534
TOC
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Analytical Chemistry
Figure 1. (a) Comparison of fluorescence intensity of Eu/Tb nanospheres with other nanospheres. (b) Fluorescence excitation (350 nm) and emission spectra (618 nm) of Eu/Tb nanospheres. 152x59mm (300 x 300 DPI)
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Analytical Chemistry
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Figure 2. (a) FTIR spectra of the Eu/Tb nanospheres, (b) 1C11 probes, (c) 2D3 probes, (d) 2–5 probes, and (e) 8# probes. (f) TEM images of the (1) Eu/Tb-nanospheres, (2) 1C11 probes, (3) 2D3 probes, (4) 2–5 probes, and (5) 8# probes. (g) EDS of spectrogram of Eu/Tb nanospheres and (h) nanosphere probes. 114x161mm (300 x 300 DPI)
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Analytical Chemistry
Figure 3. (a) Comparison of AIdnb–TRFICA and mAb–TRFICA for AFB1. (b) Comparison of AIdnb–TRFICA and mAb–TRFICA for ZEN. (c) Standard curve of AIdnb–TRFICA for AFB1. (d) Standard curve of AIdnb– TRFICA for ZEN. (e) Cross-reaction of AIdnb–TRFICA for AFB1. (f) Cross-reaction of AIdnb–TRFICA for ZEN. 114x120mm (300 x 300 DPI)
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Analytical Chemistry
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Scheme 1. Schematic illustration of AIdnb–TRFICA for dual mycotoxin. 127x95mm (300 x 300 DPI)
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