Time-Resolved Fluorescence Immunochromatographic Assay

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Article

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

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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

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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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ACS Paragon Plus Environment


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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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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.

28

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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%

228

sucrose + 1% PVPK 30 + ddH2O, were used. Therefore, the amounts of antigen on T

229

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

234

following equation:

c=

235

௡ ேఽ ௏

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where NA is the Avogadro constant, n is the number of nanospheres, and V is the

237

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

246

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

248

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

250

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

254

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

259

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

262

antibodies. The nanospheres were spherical in shape with a size of 190 nm. Figure 2f

263

(2–5) illustrates a layer of protein-wrapped nanospheres. Figure 2f (4 and 5) shows

264

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

266

between the nanospheres and nanosphere probes demonstrated that the AIdnb and

267

mAb probes were successfully conjugated. The antibodies labeled with nanospheres

268

were evidenced by comparing the EDS of nanospheres before and after conjugation,

269

as shown in Figures 2g and 2h. Characteristic peaks of C and N were obviously higher 11



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270

in the spectrum of nanospheres after conjugating with antibodies.

271

Optimizing the nanosphere probe

272

Gel electrophoresis was used to detect the size of AIdnb; it was about 15 kDa,

273

consistent with the theoretical value (Fig. S1). The concentration of AIdnb was

274

determined using the Bradford method; 2.3 mg/L of phage 2–5 and 3.5 mg/L of phage

275

8# were obtained. The amino acid sequences of phages 2–5 and 8# were analyzed

276

using the ProtParam software (http://web.expasy.org/protparam/) in the online

277

software tool ExPASy system (http://www.expasy.org/). The isoelectric point of

278

phages 8# and 2–5 was 5.81 and 6.70, respectively. Thus, the pH of the buffer

279

solution used during the conjugation should be higher than that at least.

280

The amounts for each AIdnb and mAb value were adjusted in the production of two

281

kinds of probes. Three concentrations, 10, 20, and 40 ng, of AIdnb (phages 2–5 and

282

8#) and mAb (1C11 and 2D3), were conjugated to the Eu/Tb(III) nanospheres. The

283

coupling rate (CE) was evaluated according to the following formula: CE (%)

284

=

285

antibodies, having a CE between 32% and 80.8%. As shown in Figure S2a, the

286

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

288

conjugation. A view of how the pH of boric acid buffer solution and amounts of EDC

289

affected the fluorescence intensity is given in Figure S2b and S2c. For the 1C11, 2D3,

290

and phage 8# probes, the fluorescence intensity value on the T line was almost equal

291

at pH 8.12–8.5. The phage 2–5 probe had a good fluorescence intensity at pH 8.5–9.0.

292

Different amounts, 37.5, 75, 125, and 750 ng, of EDC solution (15 mg ⋅ mL−1) were

293

examined in terms of performance to understand their remarkable effect on the

294

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

12


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295

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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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

18


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431

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17. Serge Muyldermans, M. L.. Journal of Molecular Recognition 1999,12, 131-140.

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Hammock, B. D.. Anal. Chim. Acta 2016,924, 53-59.

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X.. Talanta 2015,143, 388-393.

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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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28. Zhang, D. H.; Li, P. W.; Zhang, Q.; Zhang, W.; Huang, Y. L.; Ding, X. X.; Jiang,

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J..Analytica Chimica Acta2009, 636, 63-69.

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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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Z.. PloS one 2014,9, e85606.

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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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2015,63, 10313-8.

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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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W.. Anal Chem 2014,86, 8873-8880.

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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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Biosensors & bioelectronics 2017, 97, 59-64.

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3104-3113.

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21



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Page 22 of 32

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

22


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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

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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

24


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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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

521 522 523

Scheme 1. Schematic illustration of AIdnb–TRFICA for dual mycotoxin.

524

26


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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

535 536

28


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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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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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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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Scheme 1. Schematic illustration of AIdnb–TRFICA for dual mycotoxin. 127x95mm (300 x 300 DPI)


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Time-Resolved Fluorescence Immunochromatographic Assay

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