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Development of a Rainbow Lateral Flow Immunoassay for the Simultaneous Detection of Four Mycotoxins Astrid Foubert, Natalia V. Beloglazova, Anna Viktorovna Gordienko, Mickael D. Tessier, Emile Drijvers, Zeger Hens, and Sarah De Saeger J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04157 • Publication Date (Web): 26 Nov 2016 Downloaded from http://pubs.acs.org on November 27, 2016
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Journal of Agricultural and Food Chemistry
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Development of a Rainbow Lateral Flow Immunoassay for the Simultaneous Detection of
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Four Mycotoxins
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Astrid Foubert†,*, Natalia V. Beloglazova†, Anna Gordienko‡, Mickael D. Tessier§, Emile
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Drijvers§, Zeger Hens§, Sarah De Saeger†
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†
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Ottergemsesteenweg 460, Ghent, Belgium
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‡
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Astrakhanskaya 83, Saratov, Russia
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§
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Faculty of Pharmaceutical Sciences, Department of Bioanalysis, Laboratory of Food Analysis, Ghent University,
Chemistry Institute, Department of General Inorganic Chemistry, Chemical Institute, Saratov State University,
Faculty of Sciences, Department of Inorganic and Physical Chemistry, Ghent University, Krijgslaan 281 S3, Ghent,
Belgium
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* corresponding author. Tel.: + 32 9 264 81 33; Fax: +32 9 264 81 99
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E-mail address:
[email protected] (Astrid Foubert)
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Abstract
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A multiplex lateral flow immunoassay (LFIA) for the determination of the mycotoxins deoxynivalenol,
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zearalenone and T2/HT2-toxin in barley was developed with luminescent quantum dots (QDs) as label.
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The synthesized QDs were hydrophilized by two strategies i.e. coating with an amphiphilic polymer and
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silica. The water-soluble QDs were compared with regard to their bioconjugation with monoclonal
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antibody (mAb) and were tested on a LFIA. Silica coated QDs which contained epoxy-groups were most
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promising. Therefore, green, orange and red epoxy-functionalized silica coated QDs were conjugated
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with anti-ZEN, anti-DON and anti-T2 mAb, respectively. The LFIA was developed in accordance with the
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European Commission legal limits with cut-off limits of 1000 µg/kg, 80 µg/kg and 80 µg/kg for
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deoxynivalenol, zearalenone and T2/HT2-toxin, respectively. The LFIA gave a fast result (15 min) with a
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low false negative rate (< 5%) and the results were easy to interpret without any sophisticated
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equipment.
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Keywords: Quantum dots; Bioconjugation; Multiplex lateral flow immunoassay; Mycotoxin
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Journal of Agricultural and Food Chemistry
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Introduction
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Mycotoxins
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environmental conditions, by filamentous fungi mainly Aspergillus spp., Penicillium spp., and Fusarium
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spp. Mycotoxins are common contaminants of many cereal grains like wheat, barley, maize, and rice, and
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they can evoke a broad range of toxic properties in animals and humans and can contribute to economic
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losses.1, 2 Fusarium mycotoxins, like deoxynivalenol (DON), 1; zearalenone (ZEN), 2; T2, 3 and HT2-toxin,
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4 (T2 and HT2) (Figure 1), are widely distributed in the food chain in the EU and worldwide.3, 4 One of the
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most prevalent mycotoxin in human food is DON, also known as vomitoxin.5 DON causes a wide range of
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adverse effects upon chronic and acute exposure like genotoxicity, immune-suppression/toxicity,
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nausea, diarrhea, vomiting, anorexia and even death. Co-occurrence of DON with other Fusarium toxins,
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including ZEN is frequently observed. ZEN, also known as a myco-oestrogen, has an estrogenic action and
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is significantly toxic to the reproductive system of animals.1 T2 and HT2 also belong, like DON, to the
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trichothecenes group. Their toxic effects are, among others, a consequence of the inhibition of DNA and
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protein synthesis, with a particular effect on the digestion system (vomiting, diarrhea, haemorrhage).
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Chronic exposure to T2 also leads to immune-suppression/toxicity.5
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The importance of mycotoxin control in foodstuffs should be a major concern for food safety. Despite
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efforts to control fungal contamination, toxigenic fungi are omnipresent in nature and occur regularly in
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worldwide food supplies due to mold infestation of susceptible agricultural products.6 In order to protect
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human health from exposure to these mycotoxins the European Commission has established regulatory
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limits for DON and ZEN in cereals and cereal-based foods and feeds.7 Also, recommendations have been
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made on the presence of T2 and HT2 in cereals and cereal products.8 These increased regulatory
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requirements in food safety demand rapid, sensitive and accurate methods of analysis. In recent years
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various approaches have been proposed to perform simultaneous detection of multiple mycotoxins, for
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example, liquid chromatography - mass spectrometry (LC-MS/MS)9, enzyme linked immunosorbent assay
are
low-molecular-weight
secondary
metabolites
produced,
under
appropriate
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(ELISA)10 and different biosensors.11-14 However, all these techniques are not suitable for on-site testing
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because they require sophisticated instruments, complex operations, long analysis time and skilled
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personnel to perform the manipulations. This reinforces the need for rapid, user-friendly methods that
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can simultaneously detect multiple, co-occuring mycotoxins.
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A lateral flow immunoassay (LFIA) fulfills these criteria and would also enable quick corrective actions
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when contaminants are detected. An important part that contributes to the LFIA sensitivity is the signal
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reporter/label that enables visible detection. Until now, LFIAs for mycotoxin detection are often colloidal
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gold (CG)-based.15, 16 In this research, quantum dots (QD) were used as label because of their unique and
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stable spectroscopic properties. They are characterized by a high fluorescence quantum yield (QY),
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stability against photobleaching, and size-tunable broad absorption and narrow emission bands. In
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addition, they allow simultaneous use of multiple QDs with different spectral characteristics
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(multiplexing). It has already been shown that QDs, because of their photoluminescence brightness, are
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a more sensitive label in comparison with CG.17 However, when QDs are synthesized they are soluble in
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organic solvents like chloroform. In order to apply them in immunoassays like LFIA a hydrophilization
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step is required. This means that the QD will be covered with a hydrophilic shell.
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Two main approaches have been described in literature to transfer QDs to aqueous media, which involve
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encapsulation with amphiphilic polymers18 or ligand exchange with silica.19 Encapsulation has the
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advantage that original hydrophobic ligands remain on the QDs, while they are replaced during ligand
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exchange. Keeping the original molecules on the QD will lead to a better preservation of the QY but will
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result in larger QDs. In general, hydrophilization will lead to QDs which are stable in aqueous media and
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less toxic. It makes it possible to incorporate functional groups on the surface of the QD which can be
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used to conjugate biomolecules of interest. In particular, a silica matrix is known to give QDs with
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enhanced colloidal stability for a wide range of conditions (pH, ionic strength).19 Further, it is a relatively
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biocompatible, low cost material and the coating process is easy to control.20
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To date, QDs were seldom used in the development of LFIAs for (multi-)mycotoxin detection. There are
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reports on use of QD- based LFIAs (QD-LFIAs) for the detection of OTA21, ZEN17, AFB122 and fumonisins.23
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In other fields QD-LFIAs have been developed, for example for the detection of procalcitonine and C-
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reactive protein24 and three kinds of antibiotics.25 The latter consists of several test lines, i.e. one test line
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per analyte of interest. A multiplex QD- LFIA which consisted of one control and one test line for the
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determination of two analytes has also been developed26. However, this format has its limitations
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because the antibody binding rate will decrease if more than three antibodies are immobilized on the
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test line.
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Many publications which describe the development of QD-LFIAs use commercially available QDs and do
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not provide information about the hydrophilization shell which makes it difficult to select a proper
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conjugation strategy for the functionalization of QDs. In addition, if there is information included about
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the hydrophilization shell this information is often incomplete. Therefore, in this research QDs were
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solubilized with amphiphilic polymer and silica and compared with regard to their bioconjugation and
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applicability in a LFIA. The most optimal QDs were selected and subsequently used to develop a
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multiplex LFIA. This is the first publication which compares different hydrophilization strategies for QDs
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in combination with the development of a tricolor QD-based LFIA for the simultaneous detection of DON,
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ZEN and T2/HT2 in barley.
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Materials and methods
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Materials and reagents
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The mycotoxins ZEN and DON were purchased from Fermentek (Jerusalem, Israel). T2, HT2, poly(maleic
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anhydride-alt-1-octadecene) (PMAO, M ̴ 30 000-50 000), Tween 20 (Tween; polyoxyethylenesorbitan
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monolaurate), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide
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sodium salt (sulfo-NHS), N,N’-dicyclohexyl carbodiimide (DCC), (3-glycidyloxypropyl)trimethoxysilane 5 ACS Paragon Plus Environment
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carboxyethylsilanetrion
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(GLYMO),
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hydroxysuccinimide
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carbonyldiimidazole (CDI), bovine serum albumin (BSA), albumin from chicken egg white (OVA), casein
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sodium salt from bovine milk, phosphate buffered saline (PBS) (pH 7.4), carbonate buffered saline (CBS,
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pH 0.05 M, pH 9.6) capsules, Tris-borate-EDTA buffer, sucrose and sodium citrate were purchased from
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Sigma Aldrich (Diegem, Belgium). The QDs used were prepared at the Department of Inorganic and
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Physical Chemistry at Ghent University (Ghent, Belgium). Green emitting core/shell CdSe/ZnS QDs have
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been synthesized using a low-temperature shell growing process27. Orange and red core/shell CdSe/CdS
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QDs have been synthesized using a “flash” method28. Jeffamine M1000 (1000 g/mol) was provided by
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Huntsman (Everberg, Belgium). Water was purified using a Milli-Q Gradient System (Millipore, Brussels,
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Belgium). Methanol and the protein concentrators (9K, 20 mL) were bought from Biosolve
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(Valkenswaard, The Netherlands) and Thermo Scientific (Erembodegem, Belgium), respectively.
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Polyclonal rabbit anti-mouse immunoglobulins (2.1 g/L) were provided by DakoCytomation (Heverlee,
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Belgium). Chloroform, sodium sulfate, different nitrocellulose membranes (Hi-Flow (HF) plus 09002XSS,
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HF13502XSS, HF18002XSS, HF24002XSS), glass fiber conjugate pad and sample pad were supplied by
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Merck Millipore (Darmstadt, Germany). Membrane ‘Fusion 5’ and agarose were purchased from GE
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Healthcare (Diegem, Belgium).
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Monoclonal anti-ZEN (#1)29 and anti-DON30 antibodies were prepared at the Laboratory of Food Analysis
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at Ghent University (Ghent, Belgium). Cross-reactivity of the ZEN monoclonal antibody (mAb) was 69%
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with α-zearalenol, 42% with α-zearalanol, 22% with zearalanone and none at all (
