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Development and application of a quantitative fluorescencebased immunochromatographic assay for fumonisin B1 in maize Zhanhui Wang, Heng Li, Chenglong Li, Qing Yu, Jianzhong Shen, and Sarah De Saeger J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2014 Downloaded from http://pubs.acs.org on June 21, 2014
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 25
Journal of Agricultural and Food Chemistry
1
Development and Application of a Quantitative
2
Fluorescence-based Immunochromatographic Assay
3
for Fumonisin B1 in Maize
4
Zhanhui Wang,† Heng Li,† Chenglong Li,† Qing Yu,† Jianzhong Shen,*,† Sarah De
5
Saeger§
6 7
†
8
for Food Quality and Safety, Beijing Key Laboratory of Detection Technology for
9
Animal-Derived Food Safety , Beijing 100193, People’s Republic of China
College of Veterinary Medicine, China Agricultural University, Beijing Laboratory
10
§
11
Harelbekestraat 72, 9000 Ghent, Belgium
Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University,
12 13
* Author to whom correspondence should be addressed
14
Tel: +86-10-6273 3289
15
Fax: +86-10-6273 1032
16
E-mail: [email protected]
17 18
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
19
ABSTRACT
20
A fluorescence-based immunochromatographic assay (ICA) for fumonisin B1
21
(FB1) that employs conjugates of fluorescent microspheres and monoclonal antibodies
22
(FM-mAbs) as detection reporters is described. The ICA is based on the competitive
23
reaction between FB1-BSA (BSA, bovine serum albumin; test line) and the target FB1
24
for binding to the FM-mAb conjugates. A limit of detection (LOD) for FB1 of 0.12
25
ng/mL was obtained, with an analytical working range of 0.25-2.0 ng/mL
26
(corresponding to 250-2000 µg/kg in maize flour samples, according to the extraction
27
procedure). The recoveries of the ICA to detect FB1 in maize samples ranged from
28
91.4% to 118.2%. A quantitative comparison of the fluorescence-based ICA and
29
HPLC-MS/MS analysis of naturally contaminated maize samples indicated good
30
agreement between the two methods (r2=0.93). By replacing the target of interest, the
31
FM-based ICA can easily be extended to other chemical contaminants and thus
32
represents a versatile strategy for food safety analysis.
33
KEYWORDS: fumonisin B1, fluorescent microsphere, immunochromatographic
34
assay, maize, mycotoxins
35 36
2
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Journal of Agricultural and Food Chemistry
37
INTRODUCTION
38
Rapid assays for the detection of a variety of mycotoxins in food or feed are
39
generating ever-increasing scientific and technological interest because these assays
40
enable simple, one-step, in situ analyses.1,2 Among these rapid assays, enzyme-linked
41
immunosorbent assays (ELISAs) and immunochromatographic assays (ICAs) are two
42
well-established and accepted techniques.3-5 In practical applications, ICAs, which
43
typically use colloidal gold as the reporter, have primarily been used for the
44
qualitative detection of mycotoxins that are frequently present at relatively high
45
concentrations.6-8 Applications of ICAs have been limited by low sensitivity and poor
46
quantitative discrimination, which are intrinsically determined by the molar
47
absorption coefficient of the gold nanoparticles and the accumulating ability of the
48
analyte, respectively.9 The analysis of mycotoxins with increased sensitivity and
49
accuracy requires reporter systems that better enable high analytical sensitivity and
50
quantitative detection. Recently, a variety of reporters, including fluorescent dyes,
51
liposomes,
52
superparamagnetic nanoparticles, and fluorescent europium nanoparticles, have been
53
developed and used in an ICA format for the detection of different targets of
54
interest.10-15 However, less marked improvement in performance has been achieved,
55
and alternative reporters remain to be developed.
quantum
dots,
and
particles
such
as
silica
nanoparticles,
56
Fluorescent microspheres (FMs) are polystyrene materials that contain dyes in
57
the interior of the bead instead of merely on the surface of the bead, thereby
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
58
producing a stable configuration, high fluorescence intensity, and photostability.16,17
59
They also exhibit a narrow distribution of fluorescence intensities and sphere sizes
60
and are available in an array of colors; thus, they are potentially more accurate and
61
diverse than currently used reporters. FMs are frequently employed in suspension
62
array technologies as encoding probes, but their use as labels in the ICA format has
63
not been reported.18-20
64
Contamination of cereals and related product by mycotoxins has become an
65
increasingly serious problem. Consumers are concerned by public health-related
66
issues and show high preoccupation about the risks associated with human exposure
67
to mycotoxins.2 Fumonisins (FBs) are nephrotoxic, hepatotoxic and carcinogenic
68
mycotoxins mainly produced by Fusarium mould species (primarily F. verticillioides
69
and F. proliferatum). There are many different forms of FBs; among these, FB1
70
usually constitute about 70% of the total FBs content found in naturally contaminated
71
foods and feeds.4,7 Due to its toxic to animal and human, the FB1 has been declared by
72
the International Agency for Research on Cancer (WHO) to be a group 2B
73
carcinogen.21
74
In this work, we describe the development of a novel FM-based ICA for the
75
quantitative detection of FB1 as a model mycotoxin in maize samples. FMs were used
76
to label anti-FB1 monoclonal antibodies (mAbs) to improve the sensitivity of the
77
assay. The analytical performance of the ICA was validated by determining the levels
78
of FB1 in maize samples using an HPLC-MS/MS method as a reference method.
79
MATERIALS AND METHODS 4
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Journal of Agricultural and Food Chemistry
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Reagents. FB1 (purity ≥95.0%), FB2 (≥97.0%), and FB3 (≥97.0%) were
81
purchased from Fermentek, Ltd. (Jerusalem, Israel). Zearalenone, ochratoxin A,
82
deoxynivalenol, aflatoxin B1 and T-2 toxin were purchased from Acros Organics Co.
83
(Worcester, MA). Fluospheres carboxylate-modified microspheres, red fluorescent
84
(580/605, λex/λem), 2% solids, were purchased from Invitrogen (Carlsbad, CA). Bovine
85
serum albumin (BSA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
86
hydrochloride (EDC), and 2-(N-morpholino) ethanesulfonic acid (MES) were
87
purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were analytical
88
grade or better and were purchased from Beijing Reagent Corp. (Beijing, P.R. China).
89
The nitrocellulose filter membrane (HF13520s25) was obtained from Millipore
90
Corporation (Bedford, MA). The sample pad (CH37K) and the absorbance pad (SB08)
91
were supplied by Shanghai Liangxin Co., Ltd. (Shanghai, China). White opaque
92
96-well polystyrene microtiter plates were purchased from Costar, Inc. (Milpitas, CA).
93
The mAb 2D7 to FB1 and the coating antigen were prepared previously and were
94
purified using Protein A prior to use.22
95 96 97
MES buffer was prepared as follows: 9.76 g of MES was dissolved in 1000 mL of purified water. The pH values of MES buffer were adjusted by 1M NaOH. Apparatus. The NanoDrop ND-1000 spectrophotometer was purchased from
98
Gene Company, Ltd. (Hong Kong, P.R. China). The ZX1000 dispensing platform and
99
the CM4000 guillotine cutting module used to prepare the ICA were purchased from
100
BioDot, Inc. (Irvine, CA). The ESE Quant LFR fluorescence reader was purchased
5
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Journal of Agricultural and Food Chemistry
101
from QIAGEN (Dusseldorf, Germany). The UV spectrometer was provided by
102
Qiangyun Co. (Shanghai, P.R. China).
103
Preparation of FM-labeled FB1 mAbs. The FM-mAb conjugates were
104
prepared as follows: anti-FB1 mAb (7 µg) was dissolved in 1 mL of 50 mM MES (pH
105
6.5), and FMs (10 µL) were added dropwise. After 15 min, EDC (0.5 mg) was added
106
and maintained on a shaker for 2 h at room temperature. The pH of the reaction
107
mixture was adjusted to 6.5 using 1 M NaHCO3. To quench the reaction, 100 µL of
108
0.1 M glycine was added and incubated for 30 min. The FM-labeled mAbs were
109
obtained after centrifugation at 8,076 g for 15 min at room temperature to remove any
110
unreacted mAbs. The pellets were resuspended in 100 µL of 0.05 M PBS containing
111
0.2% BSA and 0.4% polyethylene glycol. The suspension was dispersed and blended
112
by supersonic vibration for 10 min and stored at 4 °C in the dark until use.
113
Fluorescence-based ICA. Suspensions of 0.1 mg/mL FB1-BSA and 1 mg/mL
114
goat anti-mouse IgG were dispensed onto nitrocellulose filter membranes to produce
115
the test (0.7 µL/cm) and control (0.9 µL/cm) lines, respectively. The membrane was
116
dried for 2 h at 37 °C in an air oven. The sample pad was soaked in 0.01 M PBS
117
(containing 0.05% Tween 20 and 0.05% sodium azide) and dried at 40 °C for 24 h.
118
The absorbent pad was pasted onto the top side of the backing pad by overlapping a
119
2-mm section with the membrane. The sample pad was also pasted onto the other side
120
of the backing pad by overlapping a 2-mm section with the membrane. Finally, the
121
whole assembled plate was cut into 4-mm-wide strips and stored under dry conditions
122
at room temperature. A total of 120 µL of sample solution (or standard buffer) was
6
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transferred into a 96-well microtiter plate and homogenized with 2 µL of FM-mAbs at
124
room temperature. After 5 min of shaking, the strip was added to the microwell to
125
absorb the mixture. The capillary migration process lasted approximately 15 min, and
126
the strip was then removed and fully dried for 5 min at 40 °C in an air oven prior to
127
measuring the fluorescence signal. The results of the test and control lines were either
128
qualitatively estimated by eye under a UV light or quantitatively measured by
129
measuring the signal intensities using an ESE Quant lateral flow reader set at 580 and
130
605 nm for the excitation and emission wavelengths, respectively.
131
To test the specificity of the ICA for FB2, A,
zearalenone,
T-2
and aflatoxin
toxin, deoxynivalenol
132
(DON),
133
of these mycotoxins with concentration of 0.1, 0.5, 1, 1.5, 2.0, 4.0 and 10 ng/mL were
134
prepared by diluting the corresponding stock solutions (1 mg/mL) with 0.01 M PBS.
135
The following ICA experiments were carried out in the same way as mentioned above
136
for FB1. The cross-reactivity values were calculated according to the following
137
equation:
138
ochratoxin
FB3,
B1. The standard solution
[IC50 (FB1, ng/mL)/IC50 (mycotoxins, ng/mL)] × 100%
139 140
Analysis of Maize Samples. Maize flour samples were provided by local
141
company in Belgium and stored at -20 °C. The concentration of mycotoxins was
142
determined by HPLC-MS/MS, as previously described.23 In spike and recovery
143
studies, 10 g of FB1-negative maize flour samples were spiked with FB1, which was
144
dissolved in methanol, with 500, 1000, and 1500 µg/kg. The samples were thoroughly
145
mixed and then allowed to stand at room-temperature overnight. Maize flour (1 g) 7
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Journal of Agricultural and Food Chemistry
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was vigorously mixed and extracted with 20 mL of methanol/water (4:6, v/v) for 5
147
min before ultrasonic dispersion for 5 min and centrifugation at 8,076 g for 5 min. A
148
total of 200 µL of supernatant was diluted with 800 µL of sample diluent prior to
149
analysis by the ICA as described before.
150
RESULTS AND DISCUSSION
151
Detection Principle of the FM-based ICA. In this study, the detector reagent
152
consisted of anti-FB1 mAb-functionalized FMs. The carboxyl groups of fluorescent
153
microsphere were covalently coupled to the amino group of mAb in the presence of
154
carbodiimide to insure the stability of the detector (Figure 1). The detection principle
155
of the FM-based ICA was based on the competitive binding between the FB1-BSA
156
(Test line) and the mycotoxin FB1 to combine with the limited mAbs on the
157
fluorescence reporter. As shown in Figure 1, when FB1 was present in the sample
158
solution, the FB1-FM-mAbs were formed in a microwell, diminishing the formation
159
of FB1-BSA-FM-mAbs at the test line causing its fluorescence intensity to become
160
weaker. Regardless of the presence of FB1 in the sample, secondary antibody in the
161
control line would bind to the anti-FB1 mAbs ensuring the validity of the detection.
162
According to the principle described above, the fluorescence intensity on the test line
163
would be inversely proportional to FB1 concentrations in the sample, which could be
164
used for quantitation of FB1 in samples. Quantitative analysis was realized by reading
165
the fluorescence intensities of test line with a portable strip reader.
166
FM-labeled mAbs. The activity of the FM-mAbs primarily guaranteed assay
8
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Journal of Agricultural and Food Chemistry
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speed and sensitivity; thus, we first optimized the FM-mAb preparation conditions.
168
Figure 2A shows the influence of the FM diameter on the fluorescence intensity of the
169
test line. In the presence of 300-nm FMs, the signal generation was rather slow, and it
170
took more than 30 min to accomplish the procedure. Although the 300-nm FMs
171
produced strong signals, this duration is not appropriate for a rapid assay (Figure 2A).
172
Besides, the significant residue of FM-mAb conjugates in the membrane pores could
173
affect the accurate detection of the target (Figure 2A). It took less than 10 min to
174
accomplish the procedure in the presence of 100-nm FMs; however, the signal
175
response was very weak, as shown in Figure 2A, indicating that the small-diameter
176
FMs were not appropriate for application with the ICA. The 200-nm FMs represented
177
a compromise between speed and sensitivity in the assay and were selected for further
178
experiments (Figure 2A).
179
The pH of the buffer has a great influence on the fluorescence of the FMs alone
180
and on the FM-mAb conjugates, e.g. through their dissolution and stability properties;
181
therefore, the effect of several buffers with different pH values on these FM properties
182
was evaluated. The fluorescence signal of FMs alone has been tested at pH values
183
outside 5.0-6.5, and the signal of FM-mAb conjugates at pH values between 5.0 and
184
6.5 (Figure 2B). Thus, we evaluated the effect of buffers with pH values of 5.0, 6.0
185
and 6.5 on the performance of the FM-mAb conjugates. The FM-mAb conjugates
186
clogged the membrane at pH values of 5.0 and 6.0. The pH 6.5 coupling buffer was
187
thus selected as the most suitable condition for the subsequent experiments.
188
Figure 2C shows the results of the mAb loading onto the FMs. The fluorescence
9
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189
intensity of the FMs increased as increasing amounts of mAb were added onto the
190
FMs until asymptotically approaching a maximum value. As the increase of intensity
191
is very limited for amounts of added mAb above 7 µg (Figure 2C), this quantity has
192
been selected as the most appropriate for an adequate signal sensitivity in the ICA.
193
One of the main characteristics of ICA is rapid detection, which is an important
194
advantage over other immunoassays. A time-intensity curve was constructed by
195
measuring the signal of the test line in function of incubation time. The signal
196
increased rapidly during the first 15 min, and the signal asymptotically reached a
197
maximum for longer incubation time plateaued between 15 and 30 min (Figure 2D),
198
(Figure 2D), indicating that the signal was already relatively stable for analysis at
199
15-30 min. With consideration of finding the shortest time for an optimal detection,
200
the best incubation time was considered as 15 min.
201
Analytical Performance of the ICA. The fluorescence ICA is based on
202
competitive binding of the FB1 in the samples and the FB1-BSA immobilized on the
203
test line to the FM-mAb conjugates flowing through the membrane. Analytical
204
parameters of the ICA for FB1 were obtained under the previously determined optimal
205
conditions. Concentrations of FB1 from 0-3.0 ng/mL were applied to the ICA, and the
206
corresponding inhibition fluorescence intensity of the test line was observed under an
207
ultraviolet light source or recorded by a fluorescence reader. As shown in Figure 3A,
208
the red fluorescence intensity of the test line clearly decreased as the concentration of
209
FB1 increased. The fluorescence intensity was barely detectable at 2.5 ng/mL of FB1;
210
thus, 2.5 ng/mL of FB1 was considered the cutoff value for the assay (corresponding
10
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Journal of Agricultural and Food Chemistry
211
to 25 µg/kg in maize flour samples according to the extraction procedure used in the
212
study). A calibration curve was generated using FB1 in the range of 0.25 to 2.0 ng/mL
213
(250-2000 µg/kg in maize flour samples) with an IC50 values of 1.32 ng/mL, as shown
214
in Figure 3B (r2 = 0.9949). The LOD for the quantitative detection was estimated
215
from the concentration that corresponded to the blank test line value minus 3 times the
216
standard deviation of the blank, which was calculated as 0.12 ng/mL (1.2 µg/kg in
217
maize flour samples). This LOD is lower than that of the ELISA method employing
218
the same immunoreagents (5.4 µg/kg in maize flour samples).22
219
The specificity of the ICA was determined by evaluating cross-reactivity with
220
frequently occurring mycotoxins, including FB2, FB3, T-2 toxin, deoxynivalenol
221
(DON), ochratoxin A, zearalenone, and aflatoxin B1. The ICA exhibited
222
cross-reactivities with FB2 and FB3 of 1.5% and 67.3%, respectively, and negligible
223
cross-reactivities with other mycotoxins (
Article
Development and application of a quantitative fluorescencebased immunochromatographic assay for fumonisin B1 in maize Zhanhui Wang, Heng Li, Chenglong Li, Qing Yu, Jianzhong Shen, and Sarah De Saeger J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2014 Downloaded from http://pubs.acs.org on June 21, 2014
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 25
Journal of Agricultural and Food Chemistry
1
Development and Application of a Quantitative
2
Fluorescence-based Immunochromatographic Assay
3
for Fumonisin B1 in Maize
4
Zhanhui Wang,† Heng Li,† Chenglong Li,† Qing Yu,† Jianzhong Shen,*,† Sarah De
5
Saeger§
6 7
†
8
for Food Quality and Safety, Beijing Key Laboratory of Detection Technology for
9
Animal-Derived Food Safety , Beijing 100193, People’s Republic of China
College of Veterinary Medicine, China Agricultural University, Beijing Laboratory
10
§
11
Harelbekestraat 72, 9000 Ghent, Belgium
Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University,
12 13
* Author to whom correspondence should be addressed
14
Tel: +86-10-6273 3289
15
Fax: +86-10-6273 1032
16
E-mail: [email protected]
17 18
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
19
ABSTRACT
20
A fluorescence-based immunochromatographic assay (ICA) for fumonisin B1
21
(FB1) that employs conjugates of fluorescent microspheres and monoclonal antibodies
22
(FM-mAbs) as detection reporters is described. The ICA is based on the competitive
23
reaction between FB1-BSA (BSA, bovine serum albumin; test line) and the target FB1
24
for binding to the FM-mAb conjugates. A limit of detection (LOD) for FB1 of 0.12
25
ng/mL was obtained, with an analytical working range of 0.25-2.0 ng/mL
26
(corresponding to 250-2000 µg/kg in maize flour samples, according to the extraction
27
procedure). The recoveries of the ICA to detect FB1 in maize samples ranged from
28
91.4% to 118.2%. A quantitative comparison of the fluorescence-based ICA and
29
HPLC-MS/MS analysis of naturally contaminated maize samples indicated good
30
agreement between the two methods (r2=0.93). By replacing the target of interest, the
31
FM-based ICA can easily be extended to other chemical contaminants and thus
32
represents a versatile strategy for food safety analysis.
33
KEYWORDS: fumonisin B1, fluorescent microsphere, immunochromatographic
34
assay, maize, mycotoxins
35 36
2
ACS Paragon Plus Environment
Page 2 of 25
Page 3 of 25
Journal of Agricultural and Food Chemistry
37
INTRODUCTION
38
Rapid assays for the detection of a variety of mycotoxins in food or feed are
39
generating ever-increasing scientific and technological interest because these assays
40
enable simple, one-step, in situ analyses.1,2 Among these rapid assays, enzyme-linked
41
immunosorbent assays (ELISAs) and immunochromatographic assays (ICAs) are two
42
well-established and accepted techniques.3-5 In practical applications, ICAs, which
43
typically use colloidal gold as the reporter, have primarily been used for the
44
qualitative detection of mycotoxins that are frequently present at relatively high
45
concentrations.6-8 Applications of ICAs have been limited by low sensitivity and poor
46
quantitative discrimination, which are intrinsically determined by the molar
47
absorption coefficient of the gold nanoparticles and the accumulating ability of the
48
analyte, respectively.9 The analysis of mycotoxins with increased sensitivity and
49
accuracy requires reporter systems that better enable high analytical sensitivity and
50
quantitative detection. Recently, a variety of reporters, including fluorescent dyes,
51
liposomes,
52
superparamagnetic nanoparticles, and fluorescent europium nanoparticles, have been
53
developed and used in an ICA format for the detection of different targets of
54
interest.10-15 However, less marked improvement in performance has been achieved,
55
and alternative reporters remain to be developed.
quantum
dots,
and
particles
such
as
silica
nanoparticles,
56
Fluorescent microspheres (FMs) are polystyrene materials that contain dyes in
57
the interior of the bead instead of merely on the surface of the bead, thereby
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
58
producing a stable configuration, high fluorescence intensity, and photostability.16,17
59
They also exhibit a narrow distribution of fluorescence intensities and sphere sizes
60
and are available in an array of colors; thus, they are potentially more accurate and
61
diverse than currently used reporters. FMs are frequently employed in suspension
62
array technologies as encoding probes, but their use as labels in the ICA format has
63
not been reported.18-20
64
Contamination of cereals and related product by mycotoxins has become an
65
increasingly serious problem. Consumers are concerned by public health-related
66
issues and show high preoccupation about the risks associated with human exposure
67
to mycotoxins.2 Fumonisins (FBs) are nephrotoxic, hepatotoxic and carcinogenic
68
mycotoxins mainly produced by Fusarium mould species (primarily F. verticillioides
69
and F. proliferatum). There are many different forms of FBs; among these, FB1
70
usually constitute about 70% of the total FBs content found in naturally contaminated
71
foods and feeds.4,7 Due to its toxic to animal and human, the FB1 has been declared by
72
the International Agency for Research on Cancer (WHO) to be a group 2B
73
carcinogen.21
74
In this work, we describe the development of a novel FM-based ICA for the
75
quantitative detection of FB1 as a model mycotoxin in maize samples. FMs were used
76
to label anti-FB1 monoclonal antibodies (mAbs) to improve the sensitivity of the
77
assay. The analytical performance of the ICA was validated by determining the levels
78
of FB1 in maize samples using an HPLC-MS/MS method as a reference method.
79
MATERIALS AND METHODS 4
ACS Paragon Plus Environment
Page 4 of 25
Page 5 of 25
Journal of Agricultural and Food Chemistry
80
Reagents. FB1 (purity ≥95.0%), FB2 (≥97.0%), and FB3 (≥97.0%) were
81
purchased from Fermentek, Ltd. (Jerusalem, Israel). Zearalenone, ochratoxin A,
82
deoxynivalenol, aflatoxin B1 and T-2 toxin were purchased from Acros Organics Co.
83
(Worcester, MA). Fluospheres carboxylate-modified microspheres, red fluorescent
84
(580/605, λex/λem), 2% solids, were purchased from Invitrogen (Carlsbad, CA). Bovine
85
serum albumin (BSA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
86
hydrochloride (EDC), and 2-(N-morpholino) ethanesulfonic acid (MES) were
87
purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were analytical
88
grade or better and were purchased from Beijing Reagent Corp. (Beijing, P.R. China).
89
The nitrocellulose filter membrane (HF13520s25) was obtained from Millipore
90
Corporation (Bedford, MA). The sample pad (CH37K) and the absorbance pad (SB08)
91
were supplied by Shanghai Liangxin Co., Ltd. (Shanghai, China). White opaque
92
96-well polystyrene microtiter plates were purchased from Costar, Inc. (Milpitas, CA).
93
The mAb 2D7 to FB1 and the coating antigen were prepared previously and were
94
purified using Protein A prior to use.22
95 96 97
MES buffer was prepared as follows: 9.76 g of MES was dissolved in 1000 mL of purified water. The pH values of MES buffer were adjusted by 1M NaOH. Apparatus. The NanoDrop ND-1000 spectrophotometer was purchased from
98
Gene Company, Ltd. (Hong Kong, P.R. China). The ZX1000 dispensing platform and
99
the CM4000 guillotine cutting module used to prepare the ICA were purchased from
100
BioDot, Inc. (Irvine, CA). The ESE Quant LFR fluorescence reader was purchased
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from QIAGEN (Dusseldorf, Germany). The UV spectrometer was provided by
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Qiangyun Co. (Shanghai, P.R. China).
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Preparation of FM-labeled FB1 mAbs. The FM-mAb conjugates were
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prepared as follows: anti-FB1 mAb (7 µg) was dissolved in 1 mL of 50 mM MES (pH
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6.5), and FMs (10 µL) were added dropwise. After 15 min, EDC (0.5 mg) was added
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and maintained on a shaker for 2 h at room temperature. The pH of the reaction
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mixture was adjusted to 6.5 using 1 M NaHCO3. To quench the reaction, 100 µL of
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0.1 M glycine was added and incubated for 30 min. The FM-labeled mAbs were
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obtained after centrifugation at 8,076 g for 15 min at room temperature to remove any
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unreacted mAbs. The pellets were resuspended in 100 µL of 0.05 M PBS containing
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0.2% BSA and 0.4% polyethylene glycol. The suspension was dispersed and blended
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by supersonic vibration for 10 min and stored at 4 °C in the dark until use.
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Fluorescence-based ICA. Suspensions of 0.1 mg/mL FB1-BSA and 1 mg/mL
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goat anti-mouse IgG were dispensed onto nitrocellulose filter membranes to produce
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the test (0.7 µL/cm) and control (0.9 µL/cm) lines, respectively. The membrane was
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dried for 2 h at 37 °C in an air oven. The sample pad was soaked in 0.01 M PBS
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(containing 0.05% Tween 20 and 0.05% sodium azide) and dried at 40 °C for 24 h.
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The absorbent pad was pasted onto the top side of the backing pad by overlapping a
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2-mm section with the membrane. The sample pad was also pasted onto the other side
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of the backing pad by overlapping a 2-mm section with the membrane. Finally, the
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whole assembled plate was cut into 4-mm-wide strips and stored under dry conditions
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at room temperature. A total of 120 µL of sample solution (or standard buffer) was
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transferred into a 96-well microtiter plate and homogenized with 2 µL of FM-mAbs at
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room temperature. After 5 min of shaking, the strip was added to the microwell to
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absorb the mixture. The capillary migration process lasted approximately 15 min, and
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the strip was then removed and fully dried for 5 min at 40 °C in an air oven prior to
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measuring the fluorescence signal. The results of the test and control lines were either
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qualitatively estimated by eye under a UV light or quantitatively measured by
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measuring the signal intensities using an ESE Quant lateral flow reader set at 580 and
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605 nm for the excitation and emission wavelengths, respectively.
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To test the specificity of the ICA for FB2, A,
zearalenone,
T-2
and aflatoxin
toxin, deoxynivalenol
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(DON),
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of these mycotoxins with concentration of 0.1, 0.5, 1, 1.5, 2.0, 4.0 and 10 ng/mL were
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prepared by diluting the corresponding stock solutions (1 mg/mL) with 0.01 M PBS.
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The following ICA experiments were carried out in the same way as mentioned above
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for FB1. The cross-reactivity values were calculated according to the following
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equation:
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ochratoxin
FB3,
B1. The standard solution
[IC50 (FB1, ng/mL)/IC50 (mycotoxins, ng/mL)] × 100%
139 140
Analysis of Maize Samples. Maize flour samples were provided by local
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company in Belgium and stored at -20 °C. The concentration of mycotoxins was
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determined by HPLC-MS/MS, as previously described.23 In spike and recovery
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studies, 10 g of FB1-negative maize flour samples were spiked with FB1, which was
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dissolved in methanol, with 500, 1000, and 1500 µg/kg. The samples were thoroughly
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was vigorously mixed and extracted with 20 mL of methanol/water (4:6, v/v) for 5
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min before ultrasonic dispersion for 5 min and centrifugation at 8,076 g for 5 min. A
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total of 200 µL of supernatant was diluted with 800 µL of sample diluent prior to
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analysis by the ICA as described before.
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RESULTS AND DISCUSSION
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Detection Principle of the FM-based ICA. In this study, the detector reagent
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consisted of anti-FB1 mAb-functionalized FMs. The carboxyl groups of fluorescent
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microsphere were covalently coupled to the amino group of mAb in the presence of
154
carbodiimide to insure the stability of the detector (Figure 1). The detection principle
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of the FM-based ICA was based on the competitive binding between the FB1-BSA
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(Test line) and the mycotoxin FB1 to combine with the limited mAbs on the
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fluorescence reporter. As shown in Figure 1, when FB1 was present in the sample
158
solution, the FB1-FM-mAbs were formed in a microwell, diminishing the formation
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of FB1-BSA-FM-mAbs at the test line causing its fluorescence intensity to become
160
weaker. Regardless of the presence of FB1 in the sample, secondary antibody in the
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control line would bind to the anti-FB1 mAbs ensuring the validity of the detection.
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According to the principle described above, the fluorescence intensity on the test line
163
would be inversely proportional to FB1 concentrations in the sample, which could be
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used for quantitation of FB1 in samples. Quantitative analysis was realized by reading
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the fluorescence intensities of test line with a portable strip reader.
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FM-labeled mAbs. The activity of the FM-mAbs primarily guaranteed assay
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speed and sensitivity; thus, we first optimized the FM-mAb preparation conditions.
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Figure 2A shows the influence of the FM diameter on the fluorescence intensity of the
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test line. In the presence of 300-nm FMs, the signal generation was rather slow, and it
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took more than 30 min to accomplish the procedure. Although the 300-nm FMs
171
produced strong signals, this duration is not appropriate for a rapid assay (Figure 2A).
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Besides, the significant residue of FM-mAb conjugates in the membrane pores could
173
affect the accurate detection of the target (Figure 2A). It took less than 10 min to
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accomplish the procedure in the presence of 100-nm FMs; however, the signal
175
response was very weak, as shown in Figure 2A, indicating that the small-diameter
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FMs were not appropriate for application with the ICA. The 200-nm FMs represented
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a compromise between speed and sensitivity in the assay and were selected for further
178
experiments (Figure 2A).
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The pH of the buffer has a great influence on the fluorescence of the FMs alone
180
and on the FM-mAb conjugates, e.g. through their dissolution and stability properties;
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therefore, the effect of several buffers with different pH values on these FM properties
182
was evaluated. The fluorescence signal of FMs alone has been tested at pH values
183
outside 5.0-6.5, and the signal of FM-mAb conjugates at pH values between 5.0 and
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6.5 (Figure 2B). Thus, we evaluated the effect of buffers with pH values of 5.0, 6.0
185
and 6.5 on the performance of the FM-mAb conjugates. The FM-mAb conjugates
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clogged the membrane at pH values of 5.0 and 6.0. The pH 6.5 coupling buffer was
187
thus selected as the most suitable condition for the subsequent experiments.
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Figure 2C shows the results of the mAb loading onto the FMs. The fluorescence
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intensity of the FMs increased as increasing amounts of mAb were added onto the
190
FMs until asymptotically approaching a maximum value. As the increase of intensity
191
is very limited for amounts of added mAb above 7 µg (Figure 2C), this quantity has
192
been selected as the most appropriate for an adequate signal sensitivity in the ICA.
193
One of the main characteristics of ICA is rapid detection, which is an important
194
advantage over other immunoassays. A time-intensity curve was constructed by
195
measuring the signal of the test line in function of incubation time. The signal
196
increased rapidly during the first 15 min, and the signal asymptotically reached a
197
maximum for longer incubation time plateaued between 15 and 30 min (Figure 2D),
198
(Figure 2D), indicating that the signal was already relatively stable for analysis at
199
15-30 min. With consideration of finding the shortest time for an optimal detection,
200
the best incubation time was considered as 15 min.
201
Analytical Performance of the ICA. The fluorescence ICA is based on
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competitive binding of the FB1 in the samples and the FB1-BSA immobilized on the
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test line to the FM-mAb conjugates flowing through the membrane. Analytical
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parameters of the ICA for FB1 were obtained under the previously determined optimal
205
conditions. Concentrations of FB1 from 0-3.0 ng/mL were applied to the ICA, and the
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corresponding inhibition fluorescence intensity of the test line was observed under an
207
ultraviolet light source or recorded by a fluorescence reader. As shown in Figure 3A,
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the red fluorescence intensity of the test line clearly decreased as the concentration of
209
FB1 increased. The fluorescence intensity was barely detectable at 2.5 ng/mL of FB1;
210
thus, 2.5 ng/mL of FB1 was considered the cutoff value for the assay (corresponding
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to 25 µg/kg in maize flour samples according to the extraction procedure used in the
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study). A calibration curve was generated using FB1 in the range of 0.25 to 2.0 ng/mL
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(250-2000 µg/kg in maize flour samples) with an IC50 values of 1.32 ng/mL, as shown
214
in Figure 3B (r2 = 0.9949). The LOD for the quantitative detection was estimated
215
from the concentration that corresponded to the blank test line value minus 3 times the
216
standard deviation of the blank, which was calculated as 0.12 ng/mL (1.2 µg/kg in
217
maize flour samples). This LOD is lower than that of the ELISA method employing
218
the same immunoreagents (5.4 µg/kg in maize flour samples).22
219
The specificity of the ICA was determined by evaluating cross-reactivity with
220
frequently occurring mycotoxins, including FB2, FB3, T-2 toxin, deoxynivalenol
221
(DON), ochratoxin A, zearalenone, and aflatoxin B1. The ICA exhibited
222
cross-reactivities with FB2 and FB3 of 1.5% and 67.3%, respectively, and negligible
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cross-reactivities with other mycotoxins (
