Development of a Rainbow Lateral Flow Immunoassay for the

Nov 26, 2016 - The actual encapsulation was achieved by mixing and stirring the QDs and the amphiphilic polymer in chloroform at room temperature (RT)...
2 downloads 0 Views 2MB Size
Subscriber access provided by TUFTS UNIV

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

Development of a Rainbow Lateral Flow Immunoassay for the Simultaneous Detection of

2

Four Mycotoxins

3

Astrid Foubert†,*, Natalia V. Beloglazova†, Anna Gordienko‡, Mickael D. Tessier§, Emile

4

Drijvers§, Zeger Hens§, Sarah De Saeger†

5



6

Ottergemsesteenweg 460, Ghent, Belgium

7



8

Astrakhanskaya 83, Saratov, Russia

9

§

10

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

11 12

* corresponding author. Tel.: + 32 9 264 81 33; Fax: +32 9 264 81 99

13

E-mail address: [email protected] (Astrid Foubert)

14 15

1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 38

16

Abstract

17

A multiplex lateral flow immunoassay (LFIA) for the determination of the mycotoxins deoxynivalenol,

18

zearalenone and T2/HT2-toxin in barley was developed with luminescent quantum dots (QDs) as label.

19

The synthesized QDs were hydrophilized by two strategies i.e. coating with an amphiphilic polymer and

20

silica. The water-soluble QDs were compared with regard to their bioconjugation with monoclonal

21

antibody (mAb) and were tested on a LFIA. Silica coated QDs which contained epoxy-groups were most

22

promising. Therefore, green, orange and red epoxy-functionalized silica coated QDs were conjugated

23

with anti-ZEN, anti-DON and anti-T2 mAb, respectively. The LFIA was developed in accordance with the

24

European Commission legal limits with cut-off limits of 1000 µg/kg, 80 µg/kg and 80 µg/kg for

25

deoxynivalenol, zearalenone and T2/HT2-toxin, respectively. The LFIA gave a fast result (15 min) with a

26

low false negative rate (< 5%) and the results were easy to interpret without any sophisticated

27

equipment.

28

Keywords: Quantum dots; Bioconjugation; Multiplex lateral flow immunoassay; Mycotoxin

2 ACS Paragon Plus Environment

Page 3 of 38

Journal of Agricultural and Food Chemistry

29

Introduction

30

Mycotoxins

31

environmental conditions, by filamentous fungi mainly Aspergillus spp., Penicillium spp., and Fusarium

32

spp. Mycotoxins are common contaminants of many cereal grains like wheat, barley, maize, and rice, and

33

they can evoke a broad range of toxic properties in animals and humans and can contribute to economic

34

losses.1, 2 Fusarium mycotoxins, like deoxynivalenol (DON), 1; zearalenone (ZEN), 2; T2, 3 and HT2-toxin,

35

4 (T2 and HT2) (Figure 1), are widely distributed in the food chain in the EU and worldwide.3, 4 One of the

36

most prevalent mycotoxin in human food is DON, also known as vomitoxin.5 DON causes a wide range of

37

adverse effects upon chronic and acute exposure like genotoxicity, immune-suppression/toxicity,

38

nausea, diarrhea, vomiting, anorexia and even death. Co-occurrence of DON with other Fusarium toxins,

39

including ZEN is frequently observed. ZEN, also known as a myco-oestrogen, has an estrogenic action and

40

is significantly toxic to the reproductive system of animals.1 T2 and HT2 also belong, like DON, to the

41

trichothecenes group. Their toxic effects are, among others, a consequence of the inhibition of DNA and

42

protein synthesis, with a particular effect on the digestion system (vomiting, diarrhea, haemorrhage).

43

Chronic exposure to T2 also leads to immune-suppression/toxicity.5

44

The importance of mycotoxin control in foodstuffs should be a major concern for food safety. Despite

45

efforts to control fungal contamination, toxigenic fungi are omnipresent in nature and occur regularly in

46

worldwide food supplies due to mold infestation of susceptible agricultural products.6 In order to protect

47

human health from exposure to these mycotoxins the European Commission has established regulatory

48

limits for DON and ZEN in cereals and cereal-based foods and feeds.7 Also, recommendations have been

49

made on the presence of T2 and HT2 in cereals and cereal products.8 These increased regulatory

50

requirements in food safety demand rapid, sensitive and accurate methods of analysis. In recent years

51

various approaches have been proposed to perform simultaneous detection of multiple mycotoxins, for

52

example, liquid chromatography - mass spectrometry (LC-MS/MS)9, enzyme linked immunosorbent assay

are

low-molecular-weight

secondary

metabolites

produced,

under

appropriate

3 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 38

53

(ELISA)10 and different biosensors.11-14 However, all these techniques are not suitable for on-site testing

54

because they require sophisticated instruments, complex operations, long analysis time and skilled

55

personnel to perform the manipulations. This reinforces the need for rapid, user-friendly methods that

56

can simultaneously detect multiple, co-occuring mycotoxins.

57

A lateral flow immunoassay (LFIA) fulfills these criteria and would also enable quick corrective actions

58

when contaminants are detected. An important part that contributes to the LFIA sensitivity is the signal

59

reporter/label that enables visible detection. Until now, LFIAs for mycotoxin detection are often colloidal

60

gold (CG)-based.15, 16 In this research, quantum dots (QD) were used as label because of their unique and

61

stable spectroscopic properties. They are characterized by a high fluorescence quantum yield (QY),

62

stability against photobleaching, and size-tunable broad absorption and narrow emission bands. In

63

addition, they allow simultaneous use of multiple QDs with different spectral characteristics

64

(multiplexing). It has already been shown that QDs, because of their photoluminescence brightness, are

65

a more sensitive label in comparison with CG.17 However, when QDs are synthesized they are soluble in

66

organic solvents like chloroform. In order to apply them in immunoassays like LFIA a hydrophilization

67

step is required. This means that the QD will be covered with a hydrophilic shell.

68

Two main approaches have been described in literature to transfer QDs to aqueous media, which involve

69

encapsulation with amphiphilic polymers18 or ligand exchange with silica.19 Encapsulation has the

70

advantage that original hydrophobic ligands remain on the QDs, while they are replaced during ligand

71

exchange. Keeping the original molecules on the QD will lead to a better preservation of the QY but will

72

result in larger QDs. In general, hydrophilization will lead to QDs which are stable in aqueous media and

73

less toxic. It makes it possible to incorporate functional groups on the surface of the QD which can be

74

used to conjugate biomolecules of interest. In particular, a silica matrix is known to give QDs with

75

enhanced colloidal stability for a wide range of conditions (pH, ionic strength).19 Further, it is a relatively

76

biocompatible, low cost material and the coating process is easy to control.20

4 ACS Paragon Plus Environment

Page 5 of 38

Journal of Agricultural and Food Chemistry

77

To date, QDs were seldom used in the development of LFIAs for (multi-)mycotoxin detection. There are

78

reports on use of QD- based LFIAs (QD-LFIAs) for the detection of OTA21, ZEN17, AFB122 and fumonisins.23

79

In other fields QD-LFIAs have been developed, for example for the detection of procalcitonine and C-

80

reactive protein24 and three kinds of antibiotics.25 The latter consists of several test lines, i.e. one test line

81

per analyte of interest. A multiplex QD- LFIA which consisted of one control and one test line for the

82

determination of two analytes has also been developed26. However, this format has its limitations

83

because the antibody binding rate will decrease if more than three antibodies are immobilized on the

84

test line.

85

Many publications which describe the development of QD-LFIAs use commercially available QDs and do

86

not provide information about the hydrophilization shell which makes it difficult to select a proper

87

conjugation strategy for the functionalization of QDs. In addition, if there is information included about

88

the hydrophilization shell this information is often incomplete. Therefore, in this research QDs were

89

solubilized with amphiphilic polymer and silica and compared with regard to their bioconjugation and

90

applicability in a LFIA. The most optimal QDs were selected and subsequently used to develop a

91

multiplex LFIA. This is the first publication which compares different hydrophilization strategies for QDs

92

in combination with the development of a tricolor QD-based LFIA for the simultaneous detection of DON,

93

ZEN and T2/HT2 in barley.

94

Materials and methods

95

Materials and reagents

96

The mycotoxins ZEN and DON were purchased from Fermentek (Jerusalem, Israel). T2, HT2, poly(maleic

97

anhydride-alt-1-octadecene) (PMAO, M ̴ 30 000-50 000), Tween 20 (Tween; polyoxyethylenesorbitan

98

monolaurate), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide

99

sodium salt (sulfo-NHS), N,N’-dicyclohexyl carbodiimide (DCC), (3-glycidyloxypropyl)trimethoxysilane 5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

carboxyethylsilanetrion

100

(GLYMO),

101

hydroxysuccinimide

102

carbonyldiimidazole (CDI), bovine serum albumin (BSA), albumin from chicken egg white (OVA), casein

103

sodium salt from bovine milk, phosphate buffered saline (PBS) (pH 7.4), carbonate buffered saline (CBS,

104

pH 0.05 M, pH 9.6) capsules, Tris-borate-EDTA buffer, sucrose and sodium citrate were purchased from

105

Sigma Aldrich (Diegem, Belgium). The QDs used were prepared at the Department of Inorganic and

106

Physical Chemistry at Ghent University (Ghent, Belgium). Green emitting core/shell CdSe/ZnS QDs have

107

been synthesized using a low-temperature shell growing process27. Orange and red core/shell CdSe/CdS

108

QDs have been synthesized using a “flash” method28. Jeffamine M1000 (1000 g/mol) was provided by

109

Huntsman (Everberg, Belgium). Water was purified using a Milli-Q Gradient System (Millipore, Brussels,

110

Belgium). Methanol and the protein concentrators (9K, 20 mL) were bought from Biosolve

111

(Valkenswaard, The Netherlands) and Thermo Scientific (Erembodegem, Belgium), respectively.

112

Polyclonal rabbit anti-mouse immunoglobulins (2.1 g/L) were provided by DakoCytomation (Heverlee,

113

Belgium). Chloroform, sodium sulfate, different nitrocellulose membranes (Hi-Flow (HF) plus 09002XSS,

114

HF13502XSS, HF18002XSS, HF24002XSS), glass fiber conjugate pad and sample pad were supplied by

115

Merck Millipore (Darmstadt, Germany). Membrane ‘Fusion 5’ and agarose were purchased from GE

116

Healthcare (Diegem, Belgium).

117

Monoclonal anti-ZEN (#1)29 and anti-DON30 antibodies were prepared at the Laboratory of Food Analysis

118

at Ghent University (Ghent, Belgium). Cross-reactivity of the ZEN monoclonal antibody (mAb) was 69%

119

with α-zearalenol, 42% with α-zearalanol, 22% with zearalanone and none at all (