- Email: [email protected]
Ultrasensitive immunoassays based on biotin–streptavidin amplified system for quantitative determination of family zearalenones
Accepted Manuscript Ultrasensitive immunoassays based on biotin-streptavidin amplified system for quantitative determination of family zearalenones Na Liu, Dongxia Nie, Zhiyong Zhao, Xianjun Meng, Aibo Wu PII:
S0956-7135(15)00225-X
DOI:
10.1016/j.foodcont.2015.03.049
Reference:
JFCO 4405
To appear in:
Food Control
Received Date: 12 January 2015 Revised Date:
27 March 2015
Accepted Date: 28 March 2015
Please cite this article as: Liu N., Nie D., Zhao Z., Meng X. & Wu A., Ultrasensitive immunoassays based on biotin-streptavidin amplified system for quantitative determination of family zearalenones, Food Control (2015), doi: 10.1016/j.foodcont.2015.03.049. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Ultrasensitive immunoassays based on
2
biotin-streptavidin amplified system for quantitative
3
determination of family zearalenones b,c
c
Aibo Wu
5 a
a,*
‐
Key Laboratory of Food Safety Research, Institute for Nutritional Sciences,
SC
6
c
, Dongxia Nie , Zhiyong Zhao , Xianjun Meng b,
RI PT
Na Liu
4
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294
8
Taiyuan Road, Shanghai 200031, P. R. China; Key Laboratory of Food Safety
9
Risk Assessment, Ministry of Health, Beijing, 100021, P.R. China
11 12
b
College of Food Science, Shenyang Agriculture University, 120 Dongling Road,
Shenyang 110161, Liaoning, P.R. China c
Laboratory of Quality & Safety Risk Assessment for Agro-products (Shanghai),
TE D
10
M AN U
7
13
Ministry of Agriculture, Institute for Agri-food Standards and Testing Technology,
14
Shanghai Academy of Agriculture Science, 1000 Jinqi Road, Shanghai 201403, P.R.
15
China
EP
16
* Correspondences to Aibo Wu, Key Laboratory of Food Safety Research, Institute
18
for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese
19
Academy
20
+86-21-54920296; Fax: +86-21-54922000; E-mail: [email protected]
AC C
17
of
Sciences,
Shanghai
200031,
21 22 23 24 25
1
Republic
of
China.
Phone:
ACCEPTED MANUSCRIPT 26
Abstract In this work, based on a newly obtained monoclonal antibody (MAb) against
28
zearalenone (ZEN) and biotin−streptavidin system (BSAS) for signal amplification,
29
two sensitive and rapid immunoassay formats including biotin-streptavidin amplified
30
enzyme-linked immunosorbent assay (BA-ELISA) and biotin-streptavidin amplified
31
fluorescence-linked immunosorbent assay (BA-FLISA), were developed for family
32
zearalenones (ZENs) determination. And the limits of detection (LODs) of ZEN were
33
0.02 ng mL-1 and 0.10 ng mL-1 for BA-ELISA and BA-FLISA respectively. Using the
34
BA-ELISA platform the half maximal inhibitory concentrations (IC50) were 0.18 ng
35
mL-1 for ZEN, 0.39 ng mL-1 for α-zearalenol (α-ZOL), 0.46 ng mL-1 for β-zearalenol
36
(β-ZOL), 0.30 ng mL-1 for zearalanone (ZAN), 0.30 ng mL-1 for α-zearalanol (α-ZAL),
37
and 0.73 ng mL-1 for β-zearalanol (β-ZAL). With the broad specificity, the developed
38
immunoassays could be used as sensitive and valuable tools for detection of family
39
ZENs. Additionally, the suitability of the proposed immunoassays for its application
40
to corn flour and corn based baby food has also been investigated.
41
Key words: Zearalenones (ZENs); Biotin−streptavidin system (BSAS); ELISA;
42
FLISA
45 46 47 48 49 50
SC
M AN U
TE D EP
44
AC C
43
RI PT
27
51 52 53 54 55 2
ACCEPTED MANUSCRIPT 1. Introduction
57
Zearalenone (ZEN) is an estrogenic mycotoxin as an important secondary metabolite
58
produced by several species of Fusarium molds, which is prone to infest crops,
59
including maize, rice, wheat, barley, oats, sorghum and other crops (Armando, et al.,
60
2013; Atoui, El Khoury, Kallassy, & Lebrihi, 2012). ZEN ingestion may cause central
61
precocious puberty and reproduction troubles such as infertility and lower hormone
62
levels (Aldana, Silva, Pena, Mañes V, & Lino, 2014). Furthermore it has been
63
classified in group III carcinogens by the International Agency for research on Cancer
64
(IARC, 1993, pp. 489-521). Due to the negative effect of ZEN on human health, the
65
European Union (EU) have established different maximum residue levels (MRLs) for
66
ZEN in different foodstuffs, such as 350 µg/kg in unprocessed corn, and especially
67
strict value of 20 µg/kg in processed cereal-based and corn-based baby foods for
68
infants and young children (Commission Regulation, EC No.1126/2007). Besides a
69
MRL of 60 µg/kg has been set for cereal and cereal-based products in China
70
(National food safety standard GB2761-2011). On the other hand, ZEN could be
71
metabolized into several derivates including α-ZOL, β-ZOL, ZAN, α-ZAL and β-ZAL,
72
which all belongs to family ZENs. Moreover, these metabolized compounds are also
73
harmful for human and animals due to their estrogenic activity (Belhassen, et al., 2014;
74
Välimaa, Kivistö, Leskinen, & Karp, 2010). Accordingly, monitoring the presences of
75
ZENs in food matrixes is very essential to minimize the health risk in human
76
consumption.
SC
M AN U
TE D
EP
Currently, several analytical methods have been developed for determination of
AC C
77
RI PT
56
78
ZEN and its derivates. Among these technologies, classic chromatography methods
79
are commonly used for their high accuracy such as HPLC (Ok, Choi, Kim, & Chun,
80
2014), LC-MS (Juan, Ritieni, & Mañes, 2012), and UHPLC–MS/MS (Monbaliu, Wu,
81
Zhang, Van Peteghem, & De Saeger, 2010). However, the instrumental analysis is of
82
the disadvantages of high-cost, requiring skilled operation technique, and unsuitability
83
for high-throughput screening. With the development of hybridoma technique for
84
MAb production, antibody (Ab) and its based methods have been extensively
85
developed and applied for ZEN detection, such as ELISA (Pei, Lee, Zhang, Hu, 3
ACCEPTED MANUSCRIPT Eremin, & Zhang, 2013), fluorescence immunoassays (Beloglazova, Speranskaya, De
87
Saeger, Hens, Abé, & Goryacheva, 2012), electrochemical immunoassay (Wang, et al.,
88
2013), lateral flow immunoassay (Liu, et al., 2012), and immunochip (Wang, et al.,
89
2012). Among these, ELISA is the most popular method due to its low-cost, high
90
sensitivity, easy operation and suitability for simultaneous determination a plenty of
91
samples. Considering the possible co-occurrence, a specific ELISA for group
92
detection of ZENs has been reported previously, with the broad specificity for
93
simultaneous detection of five ZENs (ZEN, α-ZOL, β-ZOL, α-ZAL and β-ZAL),
94
however the IC50 values for ZENs ranged from 100.3 ng to 131.3 ng mL-1 in buffer,
95
which were not sufficient sensitive for determination in complex matrixes, especially
96
not applicable for baby foods, where are the main focus point at the aspect of ZEN
97
monitoring (Cha, Kim, Bischoff, Kim, Son, & Kang, 2012).
M AN U
SC
RI PT
86
To improve the sensitivity of conventional ELISA, BSAS has been utilized in
99
immunoassays, which is a signal amplification system based on the high specificity
100
and strong affinity of biotin and streptavidin (STV) (Lin, et al., 2008; Pei, Cheng,
101
Wang, & Yang, 2001). More recently, BA-ELISAs have been established for trace
102
determination of antibiotics and pesticide residues in food safety with satisfactory
103
performance (Jeon, Kim, Paeng, Park, & Paeng, 2008; Wang, Zhang, Lv, Han, Zhang,
104
& Pan, 2011; Wang, Zhang, Gao, Duan, & Wang, 2010). However, very few reports
105
have been focused on BA-ELISAs for mycotoxins detection, and only one research
106
was mentioned about single ZEN detection, with a LOD of 0.35 ng mL-1 and IC50 of
107
2.071 ng mL-1 (Huang, Xu, He, Chu, Du, & Liu, 2013).
EP
AC C
108
TE D
98
Moreover, quantum dots (QDs) are widely used in various industries, which have
109
attracted great attention since their appearance due to the high photostability and
110
chemical stability (Jin & Hildebrandt, 2012; Vinayaka & Thakur, 2010). Especially,
111
QDs have been utilized as fluorescent labels for trace determination in fluorescence
112
immunoassays technology including FLISA (Trapiella-Alfonso, Costa-Fernández,
113
Pereiro, & Sanz-Medel, 2011; Yang, et al., 2014), fluorescence polarization
114
immunoassay (Tian, Zhou, Zhao, Wang, Peng, & Zhao, 2012), and fluorescence
115
resonance energy transfer (Guo, Zhang, Luo, Shen, & Sun, 2014; Xu, Xiong, Lai, Xu, 4
ACCEPTED MANUSCRIPT Li, & Xie, 2014). However, very few reports have been focused on the application of
117
QDs-based FLISA for mycotoxins determination in foodstuffs. Based on ZEN-OVA
118
labeled with QDs, Beloglazova et al developed a direct FLISA method for detection
119
of ZEN, with a LOD of 0.03 ng mL−1 and IC50 values of 0.1 ng mL−1 respectively
120
(Beloglazova, Speranskaya, De Saeger, Hens, Abé, & Goryacheva, 2012).
RI PT
116
Herein, we have applied BSAS to develop two immunoassays including BA-ELISA
122
and QDs based BA-FLISA for family ZENs detection. To the best of our knowledge,
123
there was no related report utilizing BSAS or a combination strategy of BSAS and
124
QDs in antibody-based immunoassays for determination one group of mycotoxins.
125
With these BA-immunoassays, the matrices of representative real samples of corn
126
flour and corn based baby food were applied for comparative analyses, supporting its
127
satisfactory performance for appropriate application in practical uses.
128
2. Materials and Methods
129
2.1. Reagents and materials
M AN U
SC
121
ZEN, α-ZOL, β-ZOL, ZAN, α-ZAL, β-ZAL, aflatoxin B1 (AFB1), ochratoxin A
131
(OTA), deoxynivalenol (DON) and horse radish peroxidase-labeled goat anti-mouse
132
immunoglobulin G (IgG-HRP) were purchased from sigma-Aldrich (St. Louis, MO,
133
USA).
134
(Twinsburg, Ohio, USA). Anti-ZEN MAb used in this study was prepared in our
135
previous work, which was immunized against ZEN-BSA and obtained by use of the
136
mouse hybridoma technique (Liu, et al., 2012). N-hydroxysuccinimide-biotin
137
(biotin-NHS) was obtained from the Shanghai Sangon Biological Science &
138
Technology Company (Shanghai, China). Streptavidin-HRP (STV-HRP) was
139
purchased from Beijing Bioss Biotechnology (Beijing, China). STV-QD (605nm) was
140
obtained from Wuhan Jiayuan Quantum Dots Co., Ltd (Wuhan, China). Coating
141
buffer was carbonate buffered saline (CBS, 50 mM, pH 9.6). Assay buffer was
142
phosphate buffered saline (PBS, 10 mM, pH 7.5). Washing buffer was PBS containing
143
0.05 % of Tween 20. Substrate solution A and B were prepared according to our
TE D
130
(TMB)
was
purchased
from
NBC
AC C
EP
3,3,5,5-tetramethylbenzidine
5
ACCEPTED MANUSCRIPT 144
previous work and mixed with equal proportion before use (Liu, et al., 2013).
145
Blocking buffer was 2 M sulfuric acid. Deionized water was purified by Milli-Q system, and free biotin-NHS was filtrated
147
through Amicon Ultra-0.5 mL centrifugal filters with cut off (MWCO) <10 kD
148
(Millipore, Bedford, MA, USA). Polystyrene 96-well microplates and white
149
non-transparent microplates were obtained from Costar Co. (Cambridge, MA, USA).
150
Absorbance and fluorescence signals were measured by a TECAN Infinite 200 Pro
151
microplate reader (Männedorf, Switzerland). Centrifugation was carried out in a
152
centrifuge Hereaus Multifuge 1L-R (Thermo Fisher Scientific, Spain). Shimadzu
153
LCMS-8030 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) were
154
used for verification.
155
2.2. Preparation of detection antigen ZEN-BSA
M AN U
SC
RI PT
146
ZEN-BSA was prepared according to a previous method (Burkin, Kononenko,
157
Soboleva, & Zotova, 2000) with some modifications as follows: ZEN (1.59 mg) was
158
dissolved in 300 µL of pyridine, then CMO (1.1 mg) was added. The reaction mixture
159
was incubated at 70 °C for 5 h to prepare CMO-ZEN. After evaporating to dryness by
160
vacuum drying, the residue was redissolved in 300 µL of DMF, and then 1.2 mg of
161
NHS and 4.2 mg of DCC were added. The mixed solution was incubated overnight at
162
4 °C with stirring. After the dropwise addition of the activated CMO-ZEN (150 µL)
163
into a BSA solution (33.5 mg BSA dissolved in 0.9 mL of CBS), the mixture was
164
stirred gently for 2 h at room temperature and overnight at 4 °C. The conjugate of
165
ZEN-BSA was dialyzed exhaustively by PBS at 4 °C to remove unreacted small
166
molecules.
167
2.3. Preparation and purification of biotinylated MAb
AC C
EP
TE D
156
168
The synthesis of biotinylated MAb was according to a previous report (Wang,
169
Zhang, Gao, Duan, & Wang, 2010), with minor modifications as follows: biotin-NHS
170
diluted with DMF (1 mg mL-1) and anti-ZEN MAb diluted with PBS (1 mg mL-1)
171
were mixed at a volume ratio of 1:10 (biotin-NHS/anti-ZEN MAb), and the mixture
6
ACCEPTED MANUSCRIPT 172
was incubated for 4 h at room temperature (25°C) and then kept another 12 h at 4°C.
173
Finally, the free biotin-NHS was removed by ultra filter with PBS buffer.
174
2.4. The procedures of three immunoassay formats involved
175
2.4.1. Rapid icELISA Bidimensional titration experiments were initially performed to estimate the
177
optimum concentrations of ZEN-BSA and anti-ZEN MAb. Microplates were coated
178
with an optimal dilution of ZEN-BSA in CBS for 2 h at 37 °C, then washed three
179
times, and dried, 200 µL of defatted milk (5 %, v/w) was added into each well for
180
blocking the remain binding sites, following the plates were incubated at 37 °C for 40
181
min, washed and dried as before. 50 µL of ZEN standard dilutions or sample solutions,
182
50 µL of optimized anti-ZEN MAb and 50 µL of diluted IgG-HRP were added
183
successively into each well, and then the plate was incubated for 1 h at 37 °C. After
184
three times washing, 100 µL of TMB substrate solution was added into each well.
185
Finally, the reaction was stopped 15 min later by adding 50 µL per well of 2 M
186
sulphuric acid, and the yellow colors of wells were read by microplate reader in a
187
wavelength 450 nm (Fig. A.1a).
188
2.4.2. BA-ELISA
TE D
M AN U
SC
RI PT
176
Similar bidimensional titration experiments were initially performed to estimate the
190
optimum concentrations of ZEN-BSA and biotinylated MAb. And the following
191
operation procedures were similar to those of icELISA except substitution anti-ZEN
192
MAb and IgG-HRP by biotinylated MAb and STV-HRP respectively (Fig. A.1b).
193
2.4.3. BA-FLISA
AC C
194
EP
189
Coating in white non-transparent microplates, the procedures of BA-FLISA were
195
similar to BA-ELISA except substitution STV-HRP by STV-QD, in addition the
196
microplates were directly measured by fluorescence at the excitation wavelength of
197
380nm and emission wavelength of 605nm after antigen-antibody reaction omitting
198
developing and stopping procedures (Fig. A.1c).
199
2.5. Cross-reactivity (CR) study
7
ACCEPTED MANUSCRIPT Under the optimal condition, the developed BA-ELISA was used to investigate the
201
specificity of the anti-ZEN MAb against other ZENs (α-ZOL, β-ZOL, α-ZAL, β-ZAL
202
and ZAN) and three other mycotoxins (AFB1, OTA, DON). And the CRs were
203
calculated according to the following equation: CR (%) = IC50 of ZEN / IC50 of other
204
analogues×100.
205
2.6. Sample preparation for immunoassays.
RI PT
200
Corn flour and corn based baby food samples were prepared by our previous
207
method (Liu, et al., 2012) with minor modification as follow: 5 g of sample was
208
macerated in 25 mL of a 70 % methanol solution, oscillated by vortex mixer for 30 s,
209
and then ultrasonicated for 15 min. Afterwards, the supernatant was 5-fold diluted
210
with PBS to eliminate the matrix effect, and 50 µL of the dilution was used for
211
quantitative determinations.‐ ‐‐
212
2.7. Spiked experiment and real samples analysis
M AN U
SC
206
Negative samples undetected by LC-MS/MS were spiked with ZEN at three levels
214
(2 ng mL-1, 10 ng mL-1, and 20 ng mL-1 for corn based baby food, and 2 ng mL-1, 15
215
ng mL-1, and 60 ng mL-1 for corn flour) in quadruplicate. And 26 real samples (each
216
13 samples of corn flour and corn based baby food) were purchased from local
217
supermarkets in China.
218
2.8. LC-MS/MS validation
EP
TE D
213
Samples (5 g) were extracted with 25 mL acetonitrile/water (84:16, v/v) in
220
ultrasonic bath for 1 h. After centrifugation (4500 g, 20 min), 10 mL of supernatant
221
was collected and vortexed with 5 ml of n-hexane for 3 min, and then the upper layer
222
(n-hexane) was removed. Before analysis, 1 mL of subnatant was diluted by an equal
223
volume of a methanol/water mixture (20:80, v/v).
AC C
219
224
LC-MS/MS analysis was performed on a Shimadzu LCMS-8030 triple quadruple
225
mass spectrometer equipped with an electrospray ionization source. Chromatographic
226
separation was achieved on Agilent Poroshell 120EC-C18 (100 mm ×3.0 mm, 2.7 µm)
227
with a flow rate of 0.3 mL min-1, and the column temperature was maintained at 40°C.
228
The mobile phase consisted of water containing 5 mM ammonium acetate (A) and
8
ACCEPTED MANUSCRIPT MeOH (B). The gradient procedure was set as follows: 1 min 10 % B, 14 min 90 % B,
230
16 min 90 % B, 16.5 min 10 % B, 18 min 10 % B, giving a total run time of 18 min.
231
An aliquot of 5 µL sample extract was injected into the chromatographic system. The
232
mass spectrometer was operated in the negative electrospray ionization (ESI-) modes
233
with multiple reactions monitoring (MRM) mode. Ionization source parameters were
234
set as follows: interface voltage, 3.5 kV; curved desolvation line (CDL) temperature,
235
250 °C; heatblock temperature, 400°C; collision-induced dissociation (CID) was
236
performed using argon as collision gas at a pressure of 230 kPa, and nitrogen was
237
used as nebulizing gas and drying gas with flow rates of 3.0 L/min and 15 L/min,
238
respectively. The optimal MS/MS parameters were determined (Table A.1), and Lab
239
Solutions system software (Shimadzu) was used for data acquisition and processing.
240
3. Results and discussion
241
3.1. Biotinylation of MAb
M AN U
SC
RI PT
229
Biotinylated MAb was a key material in BSAS immunoassays, which could bind to
243
SA-HRP and SA-QD. High-dosage of biotins binding to the variable region of MAb
244
might affect the combining activity of Ag and biotinylated MAb, which could lead to
245
sensitivity decrease. In this study, comparing several volume ratios of biotin-NHS and
246
MAb (1:5, 1:10, 1:20, and 1:50), the volume ratio of 1:10 (biotin-NHS/MAb) was
247
finally selected with the lowest IC50 value in BA-ELISA (data not shown), which was
248
consistent with previous report (Sai, et al., 2010).
249
3.2. Development of BA immunoassays and comparing with icELISA
250
3.2.1. Development of icELISA
EP
AC C
251
TE D
242
The tradition icELISA was improved in order to simplify operation procedures.
252
Most reported icELISA mainly had two separate procedures in operation, including
253
the competition of coating Ag, free Ag with Ab, and the binding Ab with secondary
254
Ab (Liu, Hsu, Lu, & Yu, 2013; Sheng, Jiang, De Saeger, Shen, Zhang, & Wang, 2012).
255
In this work, standard or sample solution, anit-ZEN MAb, and secondary Ab were
256
added into each well in sequence for one-step reaction. The simplified icELISA was
257
easy for operation, and saved the total detection time. Under the optimal conditions of
258
Ag (500 ng mL-1), MAb (1/32000), blocking solution (5 % defatted milk), and 9
ACCEPTED MANUSCRIPT IgG-HRP (1/5000), the standard curve of the improved icELISA for ZEN detection
260
was established (Fig. 1). The IC50 value of the improved icELISA was 0.85 ng mL-1,
261
which was more sensitive than the tradition icELISA (IC50=1.4 ng mL-1) in our
262
previous report (Liu, et al., 2012).
263
3.2.2. Development of BA-ELISA
RI PT
259
To enhance assay performance, parameters optimizing, including checkerboard
265
titration of ZEN-BSA (500 ng mL-1, 1000 ng mL-1, and 2000 ng mL-1) and dilutions
266
of biotinylated MAb (1/2000, 1/4000, 1/8000, 1/12000, 1/16000, 1/18000, 1/20000),
267
pH values of assay buffer (6, 6.5, 7.0, 7.5, 8.0 and 8.5), ionic concentrations of assay
268
buffer (5, 10, 20, 50, 100 and 200 mM), were performed according to two criteria
269
keeping the maximum absorbance (Amax) values between 0.8 and 1.5 absorption units
270
and getting the lowest IC50 value (Fig. A.2). Under the optimal conditions of coating
271
ZEN-BSA (500 ng mL-1), biotinylated anti-ZEN MAb (1/8000), STV-HRP (1/1000),
272
blocking solution (5 % defatted milk), assay buffer (pH of 7.5, 10 mM PBS), the
273
standard curve of BA-ELISA for ZEN detection was obtained (Fig. 1), and the IC50
274
and LOD were 0.18 ng mL-1 and 0.02 ng mL-1 respectively, with a working range
275
(20%–80% B/B0) of 0.05–0.63 ng mL-1, which were more sensitive than the improved
276
icELISA in this work.
277
3.2.3. Development of BA-FLISA
TE D
M AN U
SC
264
Several parameters including ZEN-BSA concentrations (500 ng mL-1, 1000 ng mL-1,
279
2000 ng mL-1), biotinylated MAb dilutions (1/1000, 1/1500, 1/2000, 1/4000, 1/8000),
280
and SA-QD dilutions (1/75, 1/100, 1/200, 1/300 and 1/400), were optimized according
281
to the ratio(
282
intensity (Fig. A.3). Considering the usage amount of SA-QDs for cost control and
283
available fluorescence intensity for accurate determination, 1000 ng mL-1 of
284
ZEN-BSA, 1/2000 dilution of biotinylated MAb, and 1/100 dilution of SA-QD were
285
finally selected (data not shown). the standard curve of BA-FLISA for ZEN detection
286
was established (Fig. 1), and the working range was between 0.31 ng mL-1 and 2.72
287
ng mL-1, with the IC50 of 0.95 ng mL-1 and LOD of 0.10 ng mL-1.
288
3.2.4. Comparison of three immunoassay formats
AC C
EP
278
20) of the positive fluorescence intensity and negative fluorescence
10
ACCEPTED MANUSCRIPT Among three immunoassay formats (BA-ELISA, BA-FLISA and icELISA), the
290
proposed BA-ELISA (IC50=0.18 ng mL-1) was more sensitive than the improved
291
icELISA (IC50=0.85 ng mL-1), and a similar result was reported previously for
292
chloramphenicol detection (Li Wang, Zhang, Gao, Duan, & Wang, 2010). In
293
BA-FLISA, the sensitivity of the BA-FLISA was sacrificed due to a higher ZEN-BSA
294
concentration (1000 ng mL-1) used for coating than two other immunoassays (500 ng
295
mL-1), with the IC50 similar to the improved icELISA and higher than BA-ELISA.
296
Briefly summarized, the sensitivities of immunoassays could be improved by
297
utilization of the BSAS. On the other hand, the procedures of enzyme labeling Ab or
298
secondary Ab might cause certain damage to the structure of the variable region of Ab
299
and affect the recognition ability of Ab and Ag. Using BSAS, HRP enzymes and QDs
300
were linked to the Ag-Ab complex through the tight binding ability of STV and biotin
301
without affecting the reaction efficiency of Ag and Ab. Moreover, the application of
302
QDs for signals amplification could omit the developing and stopping procedures of
303
traditional ELISA and shorten the total detection time, with the advantages of low
304
toxicity, good stability and unique optical properties.
305
3.3. The broad specificity for family ZENs detection
TE D
M AN U
SC
RI PT
289
The characteristic of Ab played an important role in development of immunological
307
methods, and direct determined the specificity of its based immunoassays. Using the
308
developed BA-ELISA, the specificity of the anti-ZEN MAb was evaluated by CRs
309
against other ZENs componds (ZAN, α-ZOL, β-ZOL, α-ZAL, β-ZAL) and three other
310
mycotoxins (AFB1, OTA, and DON). Under the optimal conditions for ZEN, standard
311
curves for five other ZENs were also established (Fig. 2), and corresponding IC50
312
values and CRs were calculated (Table 1). The CRs were 59.51 %, 46.74 %, 39.22 %,
313
60.50 %, and 24.68 % for ZAN, α-ZOL, β-ZOL, α-ZAL, and β-ZAL respectively,
314
with corresponding IC50 values of 0.30 ng mL-1, 0.39 ng mL-1, 0.46 ng mL-1, 0.30 ng
315
mL-1, and 0.73 ng mL-1. Besides, there were no CRs (< 0.1 %) with AFB1, OTA, and
316
DON. The IC50 values of all ZENs were between 0.18 ng mL-1 and 0.73 ng mL-1,
317
which indicated that the developed immunoassays were sensitive and effective for
318
family ZENs detection in complex matrixes. Although a previous study was reported
AC C
EP
306
11
ACCEPTED MANUSCRIPT for 5 ZENs detection based on a broad specific MAb in ELISA, with IC50 values of
320
131.3 ng mL-1 for ZEN, 121.3 ng mL-1 for α-ZOL, 109.9 ng mL-1 for β-ZOL, 115.1 ng
321
mL-1 for α-ZAL, and 100.3 ng mL-1 for β-ZAL in buffer, which were not sensitive
322
enough for quantitative determination of family ZENs in complex matrixes (Cha, Kim,
323
Bischoff, Kim, Son, & Kang, 2012).
324
3.4. The accuracies and precisions of BA-immunoassays in matrixes
RI PT
319
Matrix interference was a common problem for harmful residues determination in
326
immunoassays, which might affect the binding activity of Ag and Ab, form a weak
327
color in developing and cause false positive. Due to the frequent ZEN contamination
328
in corn products, corn flour and corn based baby food were selected as matrixes for
329
study. Existed ZEN in foodstuff could be extracted by organic solvent such as
330
methanol and acetonitrile, however high concentrations of solvents could affect the
331
activities of enzyme and Ab, resulting to sensitivity decrease in immunoassays. In
332
order to eliminate the elemental interaction, matrix dilution method was commonly
333
used as an effective tool in immunoassays. In this study, blank samples were firstly
334
extracted by 70% methanol solution, and then the extracts were 5-fold diluted by PBS.
335
Store ZEN were spiked into blank matrixes to yield a series matrix calibration
336
solutions ranging from 0.039 ng mL-1 to 20 ng mL-1, and the matrix-matched standard
337
curves were constructed by the SigmaPlot 12.0 (Four Parameter Logistic Curve) for
338
quantitative determination of ZEN in corn flour and corn based baby food, with LODs
339
all below 4 ng mL-1 (Table.A.2). Although the sensitivities of the immunoassays were
340
decreased for dilution factor, the developed BA-ELISA and BA-FLISA were sensitive
341
enough for ZEN detection in matrixes, which meet the legal requirement.
M AN U
TE D
EP
AC C
342
SC
325
Considering the MRLs of ZEN (EU commission regulation 1126/2007 and China
343
National food safety standard GB2761-2011), negative samples were fortified with
344
store ZEN standard solution at three levels (2, 15, and 60 for corn flour; and 2, 10, and
345
20 for corn based baby food), and the spiked samples were analyzed for evaluating
346
accuracies and precisions in two BA-immunoassays. As presented in Table 2, the
347
results of average recoveries for ZEN determination were all in the desirable range of
348
80%–120% in BA-ELISA and BA-FLISA, with inter-RSD and intra-RSD less than 12
ACCEPTED MANUSCRIPT 349
10% and 16% respectively, which demonstrated that both of the BA-ELISA and
350
BA-FLISA were effective tools for satisfactory determination of ZEN in real samples.
351
3.5. Application to real samples A total of 26 actual samples bought from supermarkets in China, including 13
353
samples of corn flour and 13 samples of corn based baby food, were analyzed by the
354
developed BA-immunoassays and validated by LC-MS/MS method, which were
355
presented in Table 3.
RI PT
352
For detection of corn flour samples, all 13 samples were contaminated by ZEN
357
using LC-MS/MS. Among these samples, the levels of 2 samples (2 and 7) were
358
above the MRL of China (> 60 µg/kg), especially high concentration of sample 7
359
(409.72 µg/kg) was also above the established MRL of EU (> 350 µg/kg). Besides
360
ZEN, α-ZOL and β-ZOL were co-existed in 2 samples (2 and 7), and β-ZOL was
361
presented in 4 samples (8, 10, 11, and 13) at low concentrations (<5 ng mL-1). Using
362
the BA-immunoassays, the results of all 13 samples (100 %) were found to contain
363
levels of ZEN above the LOD of the screening BA-ELISA and confirmed as
364
containing levels above the LOD by LC-MS/MS analysis. And 10/13 (76.92 %)
365
contaminated samples were found and well quantified due to the working range in
366
corn flour matrix by BA-FLISA, moreover the F/F0 of sample 8 was out of the
367
working range (80 %–90 % B/B0) for calculation and the possible value (P) was also
368
estimated by the matrix-matched standard curve of BA-FLISA in corn flour matrix.
EP
TE D
M AN U
SC
356
For detection of corn based baby food samples, using LC-MS/MS 11/13 (84.62 %)
370
samples were found to contain low levels of ZEN (<7 ng mL-1) which were far below
371
the MRL value of EU (20 µg/kg), indicating that the purchased samples were safe
372
enough for babies. According to the working range of BA-ELISA and BA-FLISA for
373
corn based baby food, 9/11(81.82%) samples were accurately quantified and the other
374
two samples with B/B0 between 80% and 90% were estimated by the BA-ELISA. In
375
addition, the possible values of 5 samples were also estimated by the BA-FLISA. To
376
some extent, most data obtained by the developed BA-immunoassays were consistent
377
with those obtained by LC-MS/MS, which demonstrated that the established methods
378
were sufficiently sensitive and reliable for rapid determination of family ZENs in corn
AC C
369
13
ACCEPTED MANUSCRIPT 379
products.
380
4. Conclusion Based on a newly obtained MAb with group specificity and effective BSAS, two
382
immunoassay formats (BA-ELISA and BA-FLISA) have been developed for family
383
ZENs detection. Moreover, the BA-ELISA was proved ultrasensitive in determination,
384
and BA-FLISA had the advantages of requiring less detection time and simplifying
385
the operation procedures. Besides the matrix effects have been studied in corn flour
386
and corn based baby food, and validation experiments in spiking and actual samples
387
were performed by the developed BA-immunoassays. As concluded, with the good
388
performance, the proposed immunoassays were simple, fast and high effective
389
methods for monitoring family ZENs in corn products, as well as other biomatrices.
390
Acknowledgements
391
This work was supported by National Basic Research Program of China (Grant
392
2013CB127801), Shanghai Municipal Commission for Science and Technology
393
(Grant 13231202800), and Jiangsu Technology Innovation Fund (SBC201210569)
M AN U
SC
RI PT
381
397 398 399 400
EP
396
AC C
395
TE D
394
401 402 403 404
14
ACCEPTED MANUSCRIPT 405
References
406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447
Aldana, J. R., Silva, L. J. G., Pena, A., Mañes V, J., & Lino, C. M. (2014). Occurrence and risk assessment of zearalenone in flours from Portuguese and Dutch markets. Food Control, 45, 51-55. Armando, M. R., Dogi, C. A., Poloni, V., Rosa, C. A. R., Dalcero, A. M., & Cavaglieri, L. R. (2013). In vitro study on the effect of Saccharomyces cerevisiae strains on growth and mycotoxin Food Microbiology, 161(3), 182-188.
RI PT
production by Aspergillus carbonarius and Fusarium graminearum. International Journal of Atoui, A., El Khoury, A., Kallassy, M., & Lebrihi, A. (2012). Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize. International Journal of Food Microbiology, 154(1–2), 59-65.
SC
Belhassen, H., Jiménez-Díaz, I., Ghali, R., Ghorbel, H., Molina-Molina, J. M., Olea, N., & Hedili, A. (2014). Validation of a UHPLC–MS/MS method for quantification of zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol and zearalanone in human urine. Journal of Chromatography B, 962, 68-74.
M AN U
Beloglazova, N. V., Speranskaya, E. S., De Saeger, S., Hens, Z., Abé, S., & Goryacheva, I. Y. (2012). Quantum dot based rapid tests for zearalenone detection. Analytical and Bioanalytical Chemistry, 403(10), 3013-3024.
Burkin, A. A., Kononenko, G. P., Soboleva, N. A., & Zotova, E. V. (2000). Preparation of conjugated antigens based on zearalenone carboxymethyloxime and their use in enzyme immunoassay. Applied Biochemistry and Microbiology, 36(3), 282-288.
Cha, S.-H., Kim, S.-H., Bischoff, K., Kim, H.-J., Son, S.-W., & Kang, H.-G. (2012). Production of a
TE D
highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay. Journal of veterinary science, 13(2), 119-125. Guo, J., Zhang, Y., Luo, Y., Shen, F., & Sun, C. (2014). Efficient fluorescence resonance energy transfer between oppositely charged CdTe quantum dots and gold nanoparticles for turn-on fluorescence detection of glyphosate. Talanta, 125, 385-392.
EP
Huang, Y., Xu, Y., He, Q., Chu, J., Du, B., & Liu, J. (2013). Determination of zearalenone in corn based on a biotin-avidin amplified enzyme-linked immunosorbent assay. Food and Agricultural Immunology, 25(2), 186-199.
AC C
Jeon, M., Kim, J., Paeng, K.-J., Park, S.-W., & Paeng, I. R. (2008). Biotin–avidin mediated competitive enzyme-linked immunosorbent assay to detect residues of tetracyclines in milk. Microchemical Journal, 88(1), 26-31.
Jin, Z., & Hildebrandt, N. (2012). Semiconductor quantum dots for in vitro diagnostics and cellular imaging. Trends in Biotechnology, 30(7), 394-403.
Juan, C., Ritieni, A., & Mañes, J. (2012). Determination of trichothecenes and zearalenones in grain cereal, flour and bread by liquid chromatography tandem mass spectrometry. Food Chemistry, 134(4), 2389-2397. Lin, Z., Wang, X., Li, Z.-J., Ren, S.-Q., Chen, G.-N., Ying, X.-T., & Lin, J.-M. (2008). Development of a sensitive, rapid, biotin–streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone. Talanta, 75(4), 965-972. Liu, B.-H., Hsu, Y.-T., Lu, C.-C., & Yu, F.-Y. (2013). Detecting aflatoxin B1 in foods and feeds by using sensitive rapid enzyme-linked immunosorbent assay and gold nanoparticle
15
ACCEPTED MANUSCRIPT immunochromatographic strip. Food Control, 30(1), 184-189. Liu, G., Han, Z., Nie, D., Yang, J., Zhao, Z., Zhang, J., Li, H., Liao, Y., Song, S., De Saeger, S., & Wu, A. (2012). Rapid and sensitive quantitation of zearalenone in food and feed by lateral flow immunoassay. Food Control, 27(1), 200-205. Liu, N., Han, Z., Lu, L., Wang, L., Ni, G., Zhao, Z., Wu, A., & Zheng, X. (2013). Development of a new rabbit monoclonal antibody and its based competitive indirect enzyme
linked
Agriculture, 93(3), 667-673.
RI PT
immunosorbent assay for rapid detection of sulfonamides. Journal of the Science of Food and Monbaliu, S., Wu, A., Zhang, D., Van Peteghem, C., & De Saeger, S. (2010). Multimycotoxin UPLC−MS/MS for Tea, Herbal Infusions and the Derived Drinkable Products. Journal of Agricultural and Food Chemistry, 58(24), 12664-12671.
Ok, H. E., Choi, S.-W., Kim, M., & Chun, H. S. (2014). HPLC and UPLC methods for the
SC
determination of zearalenone in noodles, cereal snacks and infant formula. Food Chemistry, 163, 252-257.
Pei, R., Cheng, Z., Wang, E., & Yang, X. (2001). Amplification of antigen–antibody interactions based on biotin labeled protein–streptavidin network complex using impedance spectroscopy.
M AN U
Biosensors and Bioelectronics, 16(6), 355-361.
Pei, S.-C., Lee, W.-J., Zhang, G.-P., Hu, X.-F., Eremin, S. A., & Zhang, L.-J. (2013). Development of anti-zearalenone monoclonal antibody and detection of zearalenone in corn products from China by ELISA. Food Control, 31(1), 65-70.
Sai, N., Chen, Y., Liu, N., Yu, G., Su, P., Feng, Y., Zhou, Z., Liu, X., Zhou, H., Gao, Z., & Ning, B. a. (2010). A sensitive immunoassay based on direct hapten coated format and biotin–streptavidin system for the detection of chloramphenicol. Talanta, 82(4), 1113-1121.
TE D
Sheng, Y., Jiang, W., De Saeger, S., Shen, J., Zhang, S., & Wang, Z. (2012). Development of a sensitive enzyme-linked immunosorbent assay for the detection of fumonisin B1 in maize. Toxicon, 60(7), 1245-1250.
Tian, J., Zhou, L., Zhao, Y., Wang, Y., Peng, Y., & Zhao, S. (2012). Multiplexed detection of tumor markers with multicolor quantum dots based on fluorescence polarization immunoassay.
EP
Talanta, 92, 72-77.
Trapiella-Alfonso, L., Costa-Fernández, J. M., Pereiro, R., & Sanz-Medel, A. (2011). Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk. Biosensors and Bioelectronics, 26(12), 4753-4759.
AC C
448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491
Välimaa, A.-L., Kivistö, A. T., Leskinen, P. I., & Karp, M. T. (2010). A novel biosensor for the detection of zearalenone family mycotoxins in milk. Journal of microbiological methods, 80(1), 44-48.
Vinayaka, A. C., & Thakur, M. S. (2010). Focus on quantum dots as potential fluorescent probes for monitoring food toxicants and foodborne pathogens. Analytical and Bioanalytical Chemistry, 397(4), 1445-1455. Wang, L., Zhang, L.-J., Lv, W., Han, S.-H., Zhang, F.-K., & Pan, J.-R. (2011). Determination of Organophosphorus Pesticides Based on Biotin-Avidin Enzyme-Linked Immunosorbent Assay. Chinese Journal of Analytical Chemistry, 39(3), 346-350. Wang, L., Zhang, Y., Gao, X., Duan, Z., & Wang, S. (2010). Determination of Chloramphenicol Residues
in
Milk
by
Enzyme-Linked
Immunosorbent
Assay:
Improvement
by
Biotin−Streptavidin-Amplified System. Journal of Agricultural and Food Chemistry, 58(6),
16
ACCEPTED MANUSCRIPT 3265-3270. Wang, Y.-K., Yan, Y.-X., Mao, Z.-W., Wang, H.-a., Zou, Q., Hao, Q.-W., Ji, W.-H., & Sun, J.-H. (2013). Highly sensitive electrochemical immunoassay for zearalenone in grain and grain-based food. Microchimica Acta, 180(3-4), 187-193. Wang, Y., Liu, N., Ning, B., Liu, M., Lv, Z., Sun, Z., Peng, Y., Chen, C., Li, J., & Gao, Z. (2012). Simultaneous and rapid detection of six different mycotoxins using an immunochip. Biosensors and Bioelectronics, 34(1), 44-50.
RI PT
Xu, W., Xiong, Y., Lai, W., Xu, Y., Li, C., & Xie, M. (2014). A homogeneous immunosensor for AFB1 detection based on FRET between different-sized quantum dots. Biosensors and Bioelectronics, 56, 144-150.
Yang, A., Zheng, Y., Long, C., Chen, H., Liu, B., Li, X., Yuan, J., & Cheng, F. (2014). Fluorescent immunosorbent assay for the detection of alpha lactalbumin in dairy products with monoclonal antibody bioconjugated with CdSe/ZnS quantum dots. Food Chemistry, 150,
SC
492 493 494 495 496 497 498 499 500 501 502 503 504 505
73-79.
Figures Captions
507
Fig 1. Standard curves of improved icELISA, BA-ELISA and BA-FLISA for ZEN
508
determination in PBS buffer. X axle presents logarithm of ZEN concentrations, and Y
509
axle represents the percentages of B/B0 and F/F0 (the ratios of absorbance and
510
fluorescence intensity in the presence and absence of ZEN) for icELISA/BA-ELISA
511
and BA-FLISA respectively. Each point presents the mean of three replicates, and
512
error bars represent the standard deviation.
513
Fig 2. Standard curves for detection of ZEN ( ), ZAN (▼), α-ZOL (●), β-ZOL (▲),
514
α-ZAL ( ), and β-ZAL (■) in BA-ELISA, which were constructed by detecting a
515
serial of standard solutions ranging from 0.01 to 10 ng mL-1. Each point represents the
516
mean of three replicates.
AC C
EP
TE D
M AN U
506
17
ACCEPTED MANUSCRIPT Table 1. IC50 and cross-reactivities (CRs) of the ZEN-related compounds in BA-ELISA Componds
Molecular structure
CRs (%)
0.18
100
RI PT
ZEN
IC50 (ng mL-1)
0.30
59.51
M AN U
SC
ZAN
AC C
α-ZOL
EP
β-ZAL
TE D
α-ZAL
β-ZOL
0.30
60.50
0.73
24.68
0.39
46.74
0.46
39.22
ACCEPTED MANUSCRIPT
corn based infant food
60 15 2 20 10 2
BA-FLISA
Found (ng mL-1)
Recovery (%)
Inter-RSD (%)
Intra-RSD (%)
Found (ng mL-1)
Recovery (%)
Inter-RSD (%)
Intra-RSD (%)
63.16±3.61 17.30±1.19 1.82±0.05 22.24±1.26 8.98±0.85 1.53±0.02
105.27 115.35 90.92 111.22 89.82 76.67
5.71 6.88 2.53 5.65 9.51 1.21
7.99 15.21 13.55 9.78 11.69 15.60
54.25±1.43 16.93±1.28 n/d 19.00±1.74 8.94±0.73 n/d
90.42 112.89 — 94.99 89.41 —
2.64 7.57 — 9.15 8.13 —
12.06 10.17 — 8.23 13.11 —
AC C
EP
TE D
n/d = not detected.
SC
Corn flour
BA-ELISA
M AN U
Matrixes
Spiked Con. (ng mL-1)
RI PT
Table 2. Analysis of ZEN-spiked corn flour and corn based baby food by BA-ELISA and BA-FLISA (n=4)
ACCEPTED MANUSCRIPT Table 3. Comparison of the analysis results for ZEN contamination in natural samples by the developed BA-ELISA, BA-FLISA and LC-MS/MS (n=4)
ZEN
ZEN
LC-MS/MS ZEN
AC C
α-ZOL -1
β-ZOL
(ng mL )
(ng mL )
(ng mL )
(ng mL )
(ng mL-1)
8.40 156.58 14.38 2.20 21.72 29.89 344.78 7.07 19.42 77.04 12.85 11.89 53.13 P (0.70) 4.19 n/d 4.80 1.57 4.12 2.78 1.03 1.60 3.54 0.99 P (0.79) n/d
n/d 165.89 13.67 n/d 9.45 10.48 202.73 P (4.31) 14.25 49.45 13.73 9.44 54.74 n/d P (4.86) n/d P (5.00) n/d P (3.73) P (3.18) n/d n/d P (4.24) n/d n/d n/d
2.39 130.55 9.08 1.53 8.20 15.13 409.72 7.13 23.69 53.58 11.69 11.99 56.04 0.42 3.50 n/d 3.36 0.95 6.38 2.04 1.09 1.85 3.68 0.31 0.59 n/d
n/d 1.07 n/d n/d n/d n/d 1.10 n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d
n/d 4.17 n/d n/d n/d n/d 3.62 0.29 n/d 0.51 0.21 n/d 0.24 n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d
P=possible concentration of existed ZEN; n/d = not detected.
-1
RI PT
-1
SC
-1
EP
Corn based baby food
BA-FLISA
M AN U
Corn flour
1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13
BA-ELISA
TE D
Matrices
Sample Numbers
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights Two BA-immunoassays were developed basing on a group specific MAb and BSAS.
Several assay parameters optimizing were performed.
RI PT
The developed immunoassays were sensitive for family ZENs determination.
Matrix effects of corn flour and corn based baby food were studied.
Validation experiments in spiking and actual samples were performed by the
AC C
EP
TE D
M AN U
SC
developed BA-immunoassays.
ACCEPTED MANUSCRIPT
Appendices for Ultrasensitive immunoassays based on biotin-streptavidin amplified system for quantitative determination of family
Na Liu
b,c
c
c
, Dongxia Nie , Zhiyong Zhao , Xianjun Meng b, a,*
SC
Aibo Wu a
RI PT
zearalenones
Key Laboratory of Food Safety Research, Institute for Nutritional Sciences,
M AN U
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, P. R. China; Key Laboratory of Food Safety Risk Assessment, Ministry of Health, Beijing, 100021, P.R. China b
College of Food Science, Shenyang Agriculture University, 120 Dongling Road,
Shenyang 110161, Liaoning, P.R. China
Laboratory of Quality & Safety Risk Assessment for Agro-products (Shanghai),
TE D
c
Ministry of Agriculture, Institute for Agri-food Standards and Testing Technology, Shanghai Academy of Agriculture Science, 1000 Jinqi Road, Shanghai 201403, P.R.
EP
China
AC C
* Correspondences to Aibo Wu, Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy
of
Sciences,
Shanghai
200031,
Republic
of
+86-21-54920296; Fax: +86-21-54922000; E-mail: [email protected]
China.
Phone:
ACCEPTED MANUSCRIPT A TABLE OF CONTENTS Fig. A.1 The operation schematics and principle of three immunoassays involved in this study. (a) icELISA, (b) BA-ELISA, (c) BA-FLISA.
RI PT
Fig. A.2 Parameters influences on the analytical characteristics of ZEN competitive standard curves in BA-ELISA. (●) represents Amax value in the absence of ZEN and (■) represents IC50 value. (a) Coating Ag and biotinylated MAb concentrations influences
SC
(b) pH influences (c) Ionic concentrations influences (d) Solvents influences. Data were obtained from standard curves performed in triplicate.
M AN U
Fig. A.3
Parameters optimization in the assay of BA-FLISA (a) Effect of coating Ag concentrations. (b) Effect of biotinylated MAb dilutions. (c) Effect of SA-QD dilutions. Each column presents the mean of three replicates, and error bars represent the standard deviation.
TE D
Table A.1
The optimal MS/MS parameters for ZENs determination. Table A.2
AC C
EP
Parameters of matrix-matched standard curves for ZEN determination
AC C
EP
TE D
M AN U
Fig. A.1
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Fig. A.2
AC C
EP
TE D
M AN U
SC
Fig. A.3
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Table A.1. The optimal MS/MS parameters for ZENs determination
ZEN
317.25
α-ZOL
319.3
β-ZOL
319.3
α-ZAL
321.3
β-ZAL
321.3
ZAN
319.3
product ions m/z
collision energy eV
131.3*
31
175.45 275.45* 130.28 160.3* 130.2 277.35* 303.35 277.45* 303.35 275.4* 205.35
25 30 38 30 34 22 22 22 22 20 24
RI PT
precursor ions m/z
SC
Mycotoxins
AC C
EP
TE D
M AN U
* quantifier ion
ACCEPTED MANUSCRIPT Table A.2. Parameters of matrix-matched standard curves for ZEN determination Formats
Working ranges (ng mL-1)
IC50 (ng mL-1)
LOD (ng mL-1)
BA-ELISA BA-FLISA BA-ELISA BA-FLISA
2.00–69.20 9.18–141.42 0.98–31.73 6.63–110.84
10.86 32.77 5.67 27.86
0.83 3.86 0.41 2.79
Matrixes Corn flour
AC C
EP
TE D
M AN U
SC
RI PT
Corn based baby food
S0956-7135(15)00225-X
DOI:
10.1016/j.foodcont.2015.03.049
Reference:
JFCO 4405
To appear in:
Food Control
Received Date: 12 January 2015 Revised Date:
27 March 2015
Accepted Date: 28 March 2015
Please cite this article as: Liu N., Nie D., Zhao Z., Meng X. & Wu A., Ultrasensitive immunoassays based on biotin-streptavidin amplified system for quantitative determination of family zearalenones, Food Control (2015), doi: 10.1016/j.foodcont.2015.03.049. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Ultrasensitive immunoassays based on
2
biotin-streptavidin amplified system for quantitative
3
determination of family zearalenones b,c
c
Aibo Wu
5 a
a,*
‐
Key Laboratory of Food Safety Research, Institute for Nutritional Sciences,
SC
6
c
, Dongxia Nie , Zhiyong Zhao , Xianjun Meng b,
RI PT
Na Liu
4
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294
8
Taiyuan Road, Shanghai 200031, P. R. China; Key Laboratory of Food Safety
9
Risk Assessment, Ministry of Health, Beijing, 100021, P.R. China
11 12
b
College of Food Science, Shenyang Agriculture University, 120 Dongling Road,
Shenyang 110161, Liaoning, P.R. China c
Laboratory of Quality & Safety Risk Assessment for Agro-products (Shanghai),
TE D
10
M AN U
7
13
Ministry of Agriculture, Institute for Agri-food Standards and Testing Technology,
14
Shanghai Academy of Agriculture Science, 1000 Jinqi Road, Shanghai 201403, P.R.
15
China
EP
16
* Correspondences to Aibo Wu, Key Laboratory of Food Safety Research, Institute
18
for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese
19
Academy
20
+86-21-54920296; Fax: +86-21-54922000; E-mail: [email protected]
AC C
17
of
Sciences,
Shanghai
200031,
21 22 23 24 25
1
Republic
of
China.
Phone:
ACCEPTED MANUSCRIPT 26
Abstract In this work, based on a newly obtained monoclonal antibody (MAb) against
28
zearalenone (ZEN) and biotin−streptavidin system (BSAS) for signal amplification,
29
two sensitive and rapid immunoassay formats including biotin-streptavidin amplified
30
enzyme-linked immunosorbent assay (BA-ELISA) and biotin-streptavidin amplified
31
fluorescence-linked immunosorbent assay (BA-FLISA), were developed for family
32
zearalenones (ZENs) determination. And the limits of detection (LODs) of ZEN were
33
0.02 ng mL-1 and 0.10 ng mL-1 for BA-ELISA and BA-FLISA respectively. Using the
34
BA-ELISA platform the half maximal inhibitory concentrations (IC50) were 0.18 ng
35
mL-1 for ZEN, 0.39 ng mL-1 for α-zearalenol (α-ZOL), 0.46 ng mL-1 for β-zearalenol
36
(β-ZOL), 0.30 ng mL-1 for zearalanone (ZAN), 0.30 ng mL-1 for α-zearalanol (α-ZAL),
37
and 0.73 ng mL-1 for β-zearalanol (β-ZAL). With the broad specificity, the developed
38
immunoassays could be used as sensitive and valuable tools for detection of family
39
ZENs. Additionally, the suitability of the proposed immunoassays for its application
40
to corn flour and corn based baby food has also been investigated.
41
Key words: Zearalenones (ZENs); Biotin−streptavidin system (BSAS); ELISA;
42
FLISA
45 46 47 48 49 50
SC
M AN U
TE D EP
44
AC C
43
RI PT
27
51 52 53 54 55 2
ACCEPTED MANUSCRIPT 1. Introduction
57
Zearalenone (ZEN) is an estrogenic mycotoxin as an important secondary metabolite
58
produced by several species of Fusarium molds, which is prone to infest crops,
59
including maize, rice, wheat, barley, oats, sorghum and other crops (Armando, et al.,
60
2013; Atoui, El Khoury, Kallassy, & Lebrihi, 2012). ZEN ingestion may cause central
61
precocious puberty and reproduction troubles such as infertility and lower hormone
62
levels (Aldana, Silva, Pena, Mañes V, & Lino, 2014). Furthermore it has been
63
classified in group III carcinogens by the International Agency for research on Cancer
64
(IARC, 1993, pp. 489-521). Due to the negative effect of ZEN on human health, the
65
European Union (EU) have established different maximum residue levels (MRLs) for
66
ZEN in different foodstuffs, such as 350 µg/kg in unprocessed corn, and especially
67
strict value of 20 µg/kg in processed cereal-based and corn-based baby foods for
68
infants and young children (Commission Regulation, EC No.1126/2007). Besides a
69
MRL of 60 µg/kg has been set for cereal and cereal-based products in China
70
(National food safety standard GB2761-2011). On the other hand, ZEN could be
71
metabolized into several derivates including α-ZOL, β-ZOL, ZAN, α-ZAL and β-ZAL,
72
which all belongs to family ZENs. Moreover, these metabolized compounds are also
73
harmful for human and animals due to their estrogenic activity (Belhassen, et al., 2014;
74
Välimaa, Kivistö, Leskinen, & Karp, 2010). Accordingly, monitoring the presences of
75
ZENs in food matrixes is very essential to minimize the health risk in human
76
consumption.
SC
M AN U
TE D
EP
Currently, several analytical methods have been developed for determination of
AC C
77
RI PT
56
78
ZEN and its derivates. Among these technologies, classic chromatography methods
79
are commonly used for their high accuracy such as HPLC (Ok, Choi, Kim, & Chun,
80
2014), LC-MS (Juan, Ritieni, & Mañes, 2012), and UHPLC–MS/MS (Monbaliu, Wu,
81
Zhang, Van Peteghem, & De Saeger, 2010). However, the instrumental analysis is of
82
the disadvantages of high-cost, requiring skilled operation technique, and unsuitability
83
for high-throughput screening. With the development of hybridoma technique for
84
MAb production, antibody (Ab) and its based methods have been extensively
85
developed and applied for ZEN detection, such as ELISA (Pei, Lee, Zhang, Hu, 3
ACCEPTED MANUSCRIPT Eremin, & Zhang, 2013), fluorescence immunoassays (Beloglazova, Speranskaya, De
87
Saeger, Hens, Abé, & Goryacheva, 2012), electrochemical immunoassay (Wang, et al.,
88
2013), lateral flow immunoassay (Liu, et al., 2012), and immunochip (Wang, et al.,
89
2012). Among these, ELISA is the most popular method due to its low-cost, high
90
sensitivity, easy operation and suitability for simultaneous determination a plenty of
91
samples. Considering the possible co-occurrence, a specific ELISA for group
92
detection of ZENs has been reported previously, with the broad specificity for
93
simultaneous detection of five ZENs (ZEN, α-ZOL, β-ZOL, α-ZAL and β-ZAL),
94
however the IC50 values for ZENs ranged from 100.3 ng to 131.3 ng mL-1 in buffer,
95
which were not sufficient sensitive for determination in complex matrixes, especially
96
not applicable for baby foods, where are the main focus point at the aspect of ZEN
97
monitoring (Cha, Kim, Bischoff, Kim, Son, & Kang, 2012).
M AN U
SC
RI PT
86
To improve the sensitivity of conventional ELISA, BSAS has been utilized in
99
immunoassays, which is a signal amplification system based on the high specificity
100
and strong affinity of biotin and streptavidin (STV) (Lin, et al., 2008; Pei, Cheng,
101
Wang, & Yang, 2001). More recently, BA-ELISAs have been established for trace
102
determination of antibiotics and pesticide residues in food safety with satisfactory
103
performance (Jeon, Kim, Paeng, Park, & Paeng, 2008; Wang, Zhang, Lv, Han, Zhang,
104
& Pan, 2011; Wang, Zhang, Gao, Duan, & Wang, 2010). However, very few reports
105
have been focused on BA-ELISAs for mycotoxins detection, and only one research
106
was mentioned about single ZEN detection, with a LOD of 0.35 ng mL-1 and IC50 of
107
2.071 ng mL-1 (Huang, Xu, He, Chu, Du, & Liu, 2013).
EP
AC C
108
TE D
98
Moreover, quantum dots (QDs) are widely used in various industries, which have
109
attracted great attention since their appearance due to the high photostability and
110
chemical stability (Jin & Hildebrandt, 2012; Vinayaka & Thakur, 2010). Especially,
111
QDs have been utilized as fluorescent labels for trace determination in fluorescence
112
immunoassays technology including FLISA (Trapiella-Alfonso, Costa-Fernández,
113
Pereiro, & Sanz-Medel, 2011; Yang, et al., 2014), fluorescence polarization
114
immunoassay (Tian, Zhou, Zhao, Wang, Peng, & Zhao, 2012), and fluorescence
115
resonance energy transfer (Guo, Zhang, Luo, Shen, & Sun, 2014; Xu, Xiong, Lai, Xu, 4
ACCEPTED MANUSCRIPT Li, & Xie, 2014). However, very few reports have been focused on the application of
117
QDs-based FLISA for mycotoxins determination in foodstuffs. Based on ZEN-OVA
118
labeled with QDs, Beloglazova et al developed a direct FLISA method for detection
119
of ZEN, with a LOD of 0.03 ng mL−1 and IC50 values of 0.1 ng mL−1 respectively
120
(Beloglazova, Speranskaya, De Saeger, Hens, Abé, & Goryacheva, 2012).
RI PT
116
Herein, we have applied BSAS to develop two immunoassays including BA-ELISA
122
and QDs based BA-FLISA for family ZENs detection. To the best of our knowledge,
123
there was no related report utilizing BSAS or a combination strategy of BSAS and
124
QDs in antibody-based immunoassays for determination one group of mycotoxins.
125
With these BA-immunoassays, the matrices of representative real samples of corn
126
flour and corn based baby food were applied for comparative analyses, supporting its
127
satisfactory performance for appropriate application in practical uses.
128
2. Materials and Methods
129
2.1. Reagents and materials
M AN U
SC
121
ZEN, α-ZOL, β-ZOL, ZAN, α-ZAL, β-ZAL, aflatoxin B1 (AFB1), ochratoxin A
131
(OTA), deoxynivalenol (DON) and horse radish peroxidase-labeled goat anti-mouse
132
immunoglobulin G (IgG-HRP) were purchased from sigma-Aldrich (St. Louis, MO,
133
USA).
134
(Twinsburg, Ohio, USA). Anti-ZEN MAb used in this study was prepared in our
135
previous work, which was immunized against ZEN-BSA and obtained by use of the
136
mouse hybridoma technique (Liu, et al., 2012). N-hydroxysuccinimide-biotin
137
(biotin-NHS) was obtained from the Shanghai Sangon Biological Science &
138
Technology Company (Shanghai, China). Streptavidin-HRP (STV-HRP) was
139
purchased from Beijing Bioss Biotechnology (Beijing, China). STV-QD (605nm) was
140
obtained from Wuhan Jiayuan Quantum Dots Co., Ltd (Wuhan, China). Coating
141
buffer was carbonate buffered saline (CBS, 50 mM, pH 9.6). Assay buffer was
142
phosphate buffered saline (PBS, 10 mM, pH 7.5). Washing buffer was PBS containing
143
0.05 % of Tween 20. Substrate solution A and B were prepared according to our
TE D
130
(TMB)
was
purchased
from
NBC
AC C
EP
3,3,5,5-tetramethylbenzidine
5
ACCEPTED MANUSCRIPT 144
previous work and mixed with equal proportion before use (Liu, et al., 2013).
145
Blocking buffer was 2 M sulfuric acid. Deionized water was purified by Milli-Q system, and free biotin-NHS was filtrated
147
through Amicon Ultra-0.5 mL centrifugal filters with cut off (MWCO) <10 kD
148
(Millipore, Bedford, MA, USA). Polystyrene 96-well microplates and white
149
non-transparent microplates were obtained from Costar Co. (Cambridge, MA, USA).
150
Absorbance and fluorescence signals were measured by a TECAN Infinite 200 Pro
151
microplate reader (Männedorf, Switzerland). Centrifugation was carried out in a
152
centrifuge Hereaus Multifuge 1L-R (Thermo Fisher Scientific, Spain). Shimadzu
153
LCMS-8030 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) were
154
used for verification.
155
2.2. Preparation of detection antigen ZEN-BSA
M AN U
SC
RI PT
146
ZEN-BSA was prepared according to a previous method (Burkin, Kononenko,
157
Soboleva, & Zotova, 2000) with some modifications as follows: ZEN (1.59 mg) was
158
dissolved in 300 µL of pyridine, then CMO (1.1 mg) was added. The reaction mixture
159
was incubated at 70 °C for 5 h to prepare CMO-ZEN. After evaporating to dryness by
160
vacuum drying, the residue was redissolved in 300 µL of DMF, and then 1.2 mg of
161
NHS and 4.2 mg of DCC were added. The mixed solution was incubated overnight at
162
4 °C with stirring. After the dropwise addition of the activated CMO-ZEN (150 µL)
163
into a BSA solution (33.5 mg BSA dissolved in 0.9 mL of CBS), the mixture was
164
stirred gently for 2 h at room temperature and overnight at 4 °C. The conjugate of
165
ZEN-BSA was dialyzed exhaustively by PBS at 4 °C to remove unreacted small
166
molecules.
167
2.3. Preparation and purification of biotinylated MAb
AC C
EP
TE D
156
168
The synthesis of biotinylated MAb was according to a previous report (Wang,
169
Zhang, Gao, Duan, & Wang, 2010), with minor modifications as follows: biotin-NHS
170
diluted with DMF (1 mg mL-1) and anti-ZEN MAb diluted with PBS (1 mg mL-1)
171
were mixed at a volume ratio of 1:10 (biotin-NHS/anti-ZEN MAb), and the mixture
6
ACCEPTED MANUSCRIPT 172
was incubated for 4 h at room temperature (25°C) and then kept another 12 h at 4°C.
173
Finally, the free biotin-NHS was removed by ultra filter with PBS buffer.
174
2.4. The procedures of three immunoassay formats involved
175
2.4.1. Rapid icELISA Bidimensional titration experiments were initially performed to estimate the
177
optimum concentrations of ZEN-BSA and anti-ZEN MAb. Microplates were coated
178
with an optimal dilution of ZEN-BSA in CBS for 2 h at 37 °C, then washed three
179
times, and dried, 200 µL of defatted milk (5 %, v/w) was added into each well for
180
blocking the remain binding sites, following the plates were incubated at 37 °C for 40
181
min, washed and dried as before. 50 µL of ZEN standard dilutions or sample solutions,
182
50 µL of optimized anti-ZEN MAb and 50 µL of diluted IgG-HRP were added
183
successively into each well, and then the plate was incubated for 1 h at 37 °C. After
184
three times washing, 100 µL of TMB substrate solution was added into each well.
185
Finally, the reaction was stopped 15 min later by adding 50 µL per well of 2 M
186
sulphuric acid, and the yellow colors of wells were read by microplate reader in a
187
wavelength 450 nm (Fig. A.1a).
188
2.4.2. BA-ELISA
TE D
M AN U
SC
RI PT
176
Similar bidimensional titration experiments were initially performed to estimate the
190
optimum concentrations of ZEN-BSA and biotinylated MAb. And the following
191
operation procedures were similar to those of icELISA except substitution anti-ZEN
192
MAb and IgG-HRP by biotinylated MAb and STV-HRP respectively (Fig. A.1b).
193
2.4.3. BA-FLISA
AC C
194
EP
189
Coating in white non-transparent microplates, the procedures of BA-FLISA were
195
similar to BA-ELISA except substitution STV-HRP by STV-QD, in addition the
196
microplates were directly measured by fluorescence at the excitation wavelength of
197
380nm and emission wavelength of 605nm after antigen-antibody reaction omitting
198
developing and stopping procedures (Fig. A.1c).
199
2.5. Cross-reactivity (CR) study
7
ACCEPTED MANUSCRIPT Under the optimal condition, the developed BA-ELISA was used to investigate the
201
specificity of the anti-ZEN MAb against other ZENs (α-ZOL, β-ZOL, α-ZAL, β-ZAL
202
and ZAN) and three other mycotoxins (AFB1, OTA, DON). And the CRs were
203
calculated according to the following equation: CR (%) = IC50 of ZEN / IC50 of other
204
analogues×100.
205
2.6. Sample preparation for immunoassays.
RI PT
200
Corn flour and corn based baby food samples were prepared by our previous
207
method (Liu, et al., 2012) with minor modification as follow: 5 g of sample was
208
macerated in 25 mL of a 70 % methanol solution, oscillated by vortex mixer for 30 s,
209
and then ultrasonicated for 15 min. Afterwards, the supernatant was 5-fold diluted
210
with PBS to eliminate the matrix effect, and 50 µL of the dilution was used for
211
quantitative determinations.‐ ‐‐
212
2.7. Spiked experiment and real samples analysis
M AN U
SC
206
Negative samples undetected by LC-MS/MS were spiked with ZEN at three levels
214
(2 ng mL-1, 10 ng mL-1, and 20 ng mL-1 for corn based baby food, and 2 ng mL-1, 15
215
ng mL-1, and 60 ng mL-1 for corn flour) in quadruplicate. And 26 real samples (each
216
13 samples of corn flour and corn based baby food) were purchased from local
217
supermarkets in China.
218
2.8. LC-MS/MS validation
EP
TE D
213
Samples (5 g) were extracted with 25 mL acetonitrile/water (84:16, v/v) in
220
ultrasonic bath for 1 h. After centrifugation (4500 g, 20 min), 10 mL of supernatant
221
was collected and vortexed with 5 ml of n-hexane for 3 min, and then the upper layer
222
(n-hexane) was removed. Before analysis, 1 mL of subnatant was diluted by an equal
223
volume of a methanol/water mixture (20:80, v/v).
AC C
219
224
LC-MS/MS analysis was performed on a Shimadzu LCMS-8030 triple quadruple
225
mass spectrometer equipped with an electrospray ionization source. Chromatographic
226
separation was achieved on Agilent Poroshell 120EC-C18 (100 mm ×3.0 mm, 2.7 µm)
227
with a flow rate of 0.3 mL min-1, and the column temperature was maintained at 40°C.
228
The mobile phase consisted of water containing 5 mM ammonium acetate (A) and
8
ACCEPTED MANUSCRIPT MeOH (B). The gradient procedure was set as follows: 1 min 10 % B, 14 min 90 % B,
230
16 min 90 % B, 16.5 min 10 % B, 18 min 10 % B, giving a total run time of 18 min.
231
An aliquot of 5 µL sample extract was injected into the chromatographic system. The
232
mass spectrometer was operated in the negative electrospray ionization (ESI-) modes
233
with multiple reactions monitoring (MRM) mode. Ionization source parameters were
234
set as follows: interface voltage, 3.5 kV; curved desolvation line (CDL) temperature,
235
250 °C; heatblock temperature, 400°C; collision-induced dissociation (CID) was
236
performed using argon as collision gas at a pressure of 230 kPa, and nitrogen was
237
used as nebulizing gas and drying gas with flow rates of 3.0 L/min and 15 L/min,
238
respectively. The optimal MS/MS parameters were determined (Table A.1), and Lab
239
Solutions system software (Shimadzu) was used for data acquisition and processing.
240
3. Results and discussion
241
3.1. Biotinylation of MAb
M AN U
SC
RI PT
229
Biotinylated MAb was a key material in BSAS immunoassays, which could bind to
243
SA-HRP and SA-QD. High-dosage of biotins binding to the variable region of MAb
244
might affect the combining activity of Ag and biotinylated MAb, which could lead to
245
sensitivity decrease. In this study, comparing several volume ratios of biotin-NHS and
246
MAb (1:5, 1:10, 1:20, and 1:50), the volume ratio of 1:10 (biotin-NHS/MAb) was
247
finally selected with the lowest IC50 value in BA-ELISA (data not shown), which was
248
consistent with previous report (Sai, et al., 2010).
249
3.2. Development of BA immunoassays and comparing with icELISA
250
3.2.1. Development of icELISA
EP
AC C
251
TE D
242
The tradition icELISA was improved in order to simplify operation procedures.
252
Most reported icELISA mainly had two separate procedures in operation, including
253
the competition of coating Ag, free Ag with Ab, and the binding Ab with secondary
254
Ab (Liu, Hsu, Lu, & Yu, 2013; Sheng, Jiang, De Saeger, Shen, Zhang, & Wang, 2012).
255
In this work, standard or sample solution, anit-ZEN MAb, and secondary Ab were
256
added into each well in sequence for one-step reaction. The simplified icELISA was
257
easy for operation, and saved the total detection time. Under the optimal conditions of
258
Ag (500 ng mL-1), MAb (1/32000), blocking solution (5 % defatted milk), and 9
ACCEPTED MANUSCRIPT IgG-HRP (1/5000), the standard curve of the improved icELISA for ZEN detection
260
was established (Fig. 1). The IC50 value of the improved icELISA was 0.85 ng mL-1,
261
which was more sensitive than the tradition icELISA (IC50=1.4 ng mL-1) in our
262
previous report (Liu, et al., 2012).
263
3.2.2. Development of BA-ELISA
RI PT
259
To enhance assay performance, parameters optimizing, including checkerboard
265
titration of ZEN-BSA (500 ng mL-1, 1000 ng mL-1, and 2000 ng mL-1) and dilutions
266
of biotinylated MAb (1/2000, 1/4000, 1/8000, 1/12000, 1/16000, 1/18000, 1/20000),
267
pH values of assay buffer (6, 6.5, 7.0, 7.5, 8.0 and 8.5), ionic concentrations of assay
268
buffer (5, 10, 20, 50, 100 and 200 mM), were performed according to two criteria
269
keeping the maximum absorbance (Amax) values between 0.8 and 1.5 absorption units
270
and getting the lowest IC50 value (Fig. A.2). Under the optimal conditions of coating
271
ZEN-BSA (500 ng mL-1), biotinylated anti-ZEN MAb (1/8000), STV-HRP (1/1000),
272
blocking solution (5 % defatted milk), assay buffer (pH of 7.5, 10 mM PBS), the
273
standard curve of BA-ELISA for ZEN detection was obtained (Fig. 1), and the IC50
274
and LOD were 0.18 ng mL-1 and 0.02 ng mL-1 respectively, with a working range
275
(20%–80% B/B0) of 0.05–0.63 ng mL-1, which were more sensitive than the improved
276
icELISA in this work.
277
3.2.3. Development of BA-FLISA
TE D
M AN U
SC
264
Several parameters including ZEN-BSA concentrations (500 ng mL-1, 1000 ng mL-1,
279
2000 ng mL-1), biotinylated MAb dilutions (1/1000, 1/1500, 1/2000, 1/4000, 1/8000),
280
and SA-QD dilutions (1/75, 1/100, 1/200, 1/300 and 1/400), were optimized according
281
to the ratio(
282
intensity (Fig. A.3). Considering the usage amount of SA-QDs for cost control and
283
available fluorescence intensity for accurate determination, 1000 ng mL-1 of
284
ZEN-BSA, 1/2000 dilution of biotinylated MAb, and 1/100 dilution of SA-QD were
285
finally selected (data not shown). the standard curve of BA-FLISA for ZEN detection
286
was established (Fig. 1), and the working range was between 0.31 ng mL-1 and 2.72
287
ng mL-1, with the IC50 of 0.95 ng mL-1 and LOD of 0.10 ng mL-1.
288
3.2.4. Comparison of three immunoassay formats
AC C
EP
278
20) of the positive fluorescence intensity and negative fluorescence
10
ACCEPTED MANUSCRIPT Among three immunoassay formats (BA-ELISA, BA-FLISA and icELISA), the
290
proposed BA-ELISA (IC50=0.18 ng mL-1) was more sensitive than the improved
291
icELISA (IC50=0.85 ng mL-1), and a similar result was reported previously for
292
chloramphenicol detection (Li Wang, Zhang, Gao, Duan, & Wang, 2010). In
293
BA-FLISA, the sensitivity of the BA-FLISA was sacrificed due to a higher ZEN-BSA
294
concentration (1000 ng mL-1) used for coating than two other immunoassays (500 ng
295
mL-1), with the IC50 similar to the improved icELISA and higher than BA-ELISA.
296
Briefly summarized, the sensitivities of immunoassays could be improved by
297
utilization of the BSAS. On the other hand, the procedures of enzyme labeling Ab or
298
secondary Ab might cause certain damage to the structure of the variable region of Ab
299
and affect the recognition ability of Ab and Ag. Using BSAS, HRP enzymes and QDs
300
were linked to the Ag-Ab complex through the tight binding ability of STV and biotin
301
without affecting the reaction efficiency of Ag and Ab. Moreover, the application of
302
QDs for signals amplification could omit the developing and stopping procedures of
303
traditional ELISA and shorten the total detection time, with the advantages of low
304
toxicity, good stability and unique optical properties.
305
3.3. The broad specificity for family ZENs detection
TE D
M AN U
SC
RI PT
289
The characteristic of Ab played an important role in development of immunological
307
methods, and direct determined the specificity of its based immunoassays. Using the
308
developed BA-ELISA, the specificity of the anti-ZEN MAb was evaluated by CRs
309
against other ZENs componds (ZAN, α-ZOL, β-ZOL, α-ZAL, β-ZAL) and three other
310
mycotoxins (AFB1, OTA, and DON). Under the optimal conditions for ZEN, standard
311
curves for five other ZENs were also established (Fig. 2), and corresponding IC50
312
values and CRs were calculated (Table 1). The CRs were 59.51 %, 46.74 %, 39.22 %,
313
60.50 %, and 24.68 % for ZAN, α-ZOL, β-ZOL, α-ZAL, and β-ZAL respectively,
314
with corresponding IC50 values of 0.30 ng mL-1, 0.39 ng mL-1, 0.46 ng mL-1, 0.30 ng
315
mL-1, and 0.73 ng mL-1. Besides, there were no CRs (< 0.1 %) with AFB1, OTA, and
316
DON. The IC50 values of all ZENs were between 0.18 ng mL-1 and 0.73 ng mL-1,
317
which indicated that the developed immunoassays were sensitive and effective for
318
family ZENs detection in complex matrixes. Although a previous study was reported
AC C
EP
306
11
ACCEPTED MANUSCRIPT for 5 ZENs detection based on a broad specific MAb in ELISA, with IC50 values of
320
131.3 ng mL-1 for ZEN, 121.3 ng mL-1 for α-ZOL, 109.9 ng mL-1 for β-ZOL, 115.1 ng
321
mL-1 for α-ZAL, and 100.3 ng mL-1 for β-ZAL in buffer, which were not sensitive
322
enough for quantitative determination of family ZENs in complex matrixes (Cha, Kim,
323
Bischoff, Kim, Son, & Kang, 2012).
324
3.4. The accuracies and precisions of BA-immunoassays in matrixes
RI PT
319
Matrix interference was a common problem for harmful residues determination in
326
immunoassays, which might affect the binding activity of Ag and Ab, form a weak
327
color in developing and cause false positive. Due to the frequent ZEN contamination
328
in corn products, corn flour and corn based baby food were selected as matrixes for
329
study. Existed ZEN in foodstuff could be extracted by organic solvent such as
330
methanol and acetonitrile, however high concentrations of solvents could affect the
331
activities of enzyme and Ab, resulting to sensitivity decrease in immunoassays. In
332
order to eliminate the elemental interaction, matrix dilution method was commonly
333
used as an effective tool in immunoassays. In this study, blank samples were firstly
334
extracted by 70% methanol solution, and then the extracts were 5-fold diluted by PBS.
335
Store ZEN were spiked into blank matrixes to yield a series matrix calibration
336
solutions ranging from 0.039 ng mL-1 to 20 ng mL-1, and the matrix-matched standard
337
curves were constructed by the SigmaPlot 12.0 (Four Parameter Logistic Curve) for
338
quantitative determination of ZEN in corn flour and corn based baby food, with LODs
339
all below 4 ng mL-1 (Table.A.2). Although the sensitivities of the immunoassays were
340
decreased for dilution factor, the developed BA-ELISA and BA-FLISA were sensitive
341
enough for ZEN detection in matrixes, which meet the legal requirement.
M AN U
TE D
EP
AC C
342
SC
325
Considering the MRLs of ZEN (EU commission regulation 1126/2007 and China
343
National food safety standard GB2761-2011), negative samples were fortified with
344
store ZEN standard solution at three levels (2, 15, and 60 for corn flour; and 2, 10, and
345
20 for corn based baby food), and the spiked samples were analyzed for evaluating
346
accuracies and precisions in two BA-immunoassays. As presented in Table 2, the
347
results of average recoveries for ZEN determination were all in the desirable range of
348
80%–120% in BA-ELISA and BA-FLISA, with inter-RSD and intra-RSD less than 12
ACCEPTED MANUSCRIPT 349
10% and 16% respectively, which demonstrated that both of the BA-ELISA and
350
BA-FLISA were effective tools for satisfactory determination of ZEN in real samples.
351
3.5. Application to real samples A total of 26 actual samples bought from supermarkets in China, including 13
353
samples of corn flour and 13 samples of corn based baby food, were analyzed by the
354
developed BA-immunoassays and validated by LC-MS/MS method, which were
355
presented in Table 3.
RI PT
352
For detection of corn flour samples, all 13 samples were contaminated by ZEN
357
using LC-MS/MS. Among these samples, the levels of 2 samples (2 and 7) were
358
above the MRL of China (> 60 µg/kg), especially high concentration of sample 7
359
(409.72 µg/kg) was also above the established MRL of EU (> 350 µg/kg). Besides
360
ZEN, α-ZOL and β-ZOL were co-existed in 2 samples (2 and 7), and β-ZOL was
361
presented in 4 samples (8, 10, 11, and 13) at low concentrations (<5 ng mL-1). Using
362
the BA-immunoassays, the results of all 13 samples (100 %) were found to contain
363
levels of ZEN above the LOD of the screening BA-ELISA and confirmed as
364
containing levels above the LOD by LC-MS/MS analysis. And 10/13 (76.92 %)
365
contaminated samples were found and well quantified due to the working range in
366
corn flour matrix by BA-FLISA, moreover the F/F0 of sample 8 was out of the
367
working range (80 %–90 % B/B0) for calculation and the possible value (P) was also
368
estimated by the matrix-matched standard curve of BA-FLISA in corn flour matrix.
EP
TE D
M AN U
SC
356
For detection of corn based baby food samples, using LC-MS/MS 11/13 (84.62 %)
370
samples were found to contain low levels of ZEN (<7 ng mL-1) which were far below
371
the MRL value of EU (20 µg/kg), indicating that the purchased samples were safe
372
enough for babies. According to the working range of BA-ELISA and BA-FLISA for
373
corn based baby food, 9/11(81.82%) samples were accurately quantified and the other
374
two samples with B/B0 between 80% and 90% were estimated by the BA-ELISA. In
375
addition, the possible values of 5 samples were also estimated by the BA-FLISA. To
376
some extent, most data obtained by the developed BA-immunoassays were consistent
377
with those obtained by LC-MS/MS, which demonstrated that the established methods
378
were sufficiently sensitive and reliable for rapid determination of family ZENs in corn
AC C
369
13
ACCEPTED MANUSCRIPT 379
products.
380
4. Conclusion Based on a newly obtained MAb with group specificity and effective BSAS, two
382
immunoassay formats (BA-ELISA and BA-FLISA) have been developed for family
383
ZENs detection. Moreover, the BA-ELISA was proved ultrasensitive in determination,
384
and BA-FLISA had the advantages of requiring less detection time and simplifying
385
the operation procedures. Besides the matrix effects have been studied in corn flour
386
and corn based baby food, and validation experiments in spiking and actual samples
387
were performed by the developed BA-immunoassays. As concluded, with the good
388
performance, the proposed immunoassays were simple, fast and high effective
389
methods for monitoring family ZENs in corn products, as well as other biomatrices.
390
Acknowledgements
391
This work was supported by National Basic Research Program of China (Grant
392
2013CB127801), Shanghai Municipal Commission for Science and Technology
393
(Grant 13231202800), and Jiangsu Technology Innovation Fund (SBC201210569)
M AN U
SC
RI PT
381
397 398 399 400
EP
396
AC C
395
TE D
394
401 402 403 404
14
ACCEPTED MANUSCRIPT 405
References
406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447
Aldana, J. R., Silva, L. J. G., Pena, A., Mañes V, J., & Lino, C. M. (2014). Occurrence and risk assessment of zearalenone in flours from Portuguese and Dutch markets. Food Control, 45, 51-55. Armando, M. R., Dogi, C. A., Poloni, V., Rosa, C. A. R., Dalcero, A. M., & Cavaglieri, L. R. (2013). In vitro study on the effect of Saccharomyces cerevisiae strains on growth and mycotoxin Food Microbiology, 161(3), 182-188.
RI PT
production by Aspergillus carbonarius and Fusarium graminearum. International Journal of Atoui, A., El Khoury, A., Kallassy, M., & Lebrihi, A. (2012). Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize. International Journal of Food Microbiology, 154(1–2), 59-65.
SC
Belhassen, H., Jiménez-Díaz, I., Ghali, R., Ghorbel, H., Molina-Molina, J. M., Olea, N., & Hedili, A. (2014). Validation of a UHPLC–MS/MS method for quantification of zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol and zearalanone in human urine. Journal of Chromatography B, 962, 68-74.
M AN U
Beloglazova, N. V., Speranskaya, E. S., De Saeger, S., Hens, Z., Abé, S., & Goryacheva, I. Y. (2012). Quantum dot based rapid tests for zearalenone detection. Analytical and Bioanalytical Chemistry, 403(10), 3013-3024.
Burkin, A. A., Kononenko, G. P., Soboleva, N. A., & Zotova, E. V. (2000). Preparation of conjugated antigens based on zearalenone carboxymethyloxime and their use in enzyme immunoassay. Applied Biochemistry and Microbiology, 36(3), 282-288.
Cha, S.-H., Kim, S.-H., Bischoff, K., Kim, H.-J., Son, S.-W., & Kang, H.-G. (2012). Production of a
TE D
highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay. Journal of veterinary science, 13(2), 119-125. Guo, J., Zhang, Y., Luo, Y., Shen, F., & Sun, C. (2014). Efficient fluorescence resonance energy transfer between oppositely charged CdTe quantum dots and gold nanoparticles for turn-on fluorescence detection of glyphosate. Talanta, 125, 385-392.
EP
Huang, Y., Xu, Y., He, Q., Chu, J., Du, B., & Liu, J. (2013). Determination of zearalenone in corn based on a biotin-avidin amplified enzyme-linked immunosorbent assay. Food and Agricultural Immunology, 25(2), 186-199.
AC C
Jeon, M., Kim, J., Paeng, K.-J., Park, S.-W., & Paeng, I. R. (2008). Biotin–avidin mediated competitive enzyme-linked immunosorbent assay to detect residues of tetracyclines in milk. Microchemical Journal, 88(1), 26-31.
Jin, Z., & Hildebrandt, N. (2012). Semiconductor quantum dots for in vitro diagnostics and cellular imaging. Trends in Biotechnology, 30(7), 394-403.
Juan, C., Ritieni, A., & Mañes, J. (2012). Determination of trichothecenes and zearalenones in grain cereal, flour and bread by liquid chromatography tandem mass spectrometry. Food Chemistry, 134(4), 2389-2397. Lin, Z., Wang, X., Li, Z.-J., Ren, S.-Q., Chen, G.-N., Ying, X.-T., & Lin, J.-M. (2008). Development of a sensitive, rapid, biotin–streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone. Talanta, 75(4), 965-972. Liu, B.-H., Hsu, Y.-T., Lu, C.-C., & Yu, F.-Y. (2013). Detecting aflatoxin B1 in foods and feeds by using sensitive rapid enzyme-linked immunosorbent assay and gold nanoparticle
15
ACCEPTED MANUSCRIPT immunochromatographic strip. Food Control, 30(1), 184-189. Liu, G., Han, Z., Nie, D., Yang, J., Zhao, Z., Zhang, J., Li, H., Liao, Y., Song, S., De Saeger, S., & Wu, A. (2012). Rapid and sensitive quantitation of zearalenone in food and feed by lateral flow immunoassay. Food Control, 27(1), 200-205. Liu, N., Han, Z., Lu, L., Wang, L., Ni, G., Zhao, Z., Wu, A., & Zheng, X. (2013). Development of a new rabbit monoclonal antibody and its based competitive indirect enzyme
linked
Agriculture, 93(3), 667-673.
RI PT
immunosorbent assay for rapid detection of sulfonamides. Journal of the Science of Food and Monbaliu, S., Wu, A., Zhang, D., Van Peteghem, C., & De Saeger, S. (2010). Multimycotoxin UPLC−MS/MS for Tea, Herbal Infusions and the Derived Drinkable Products. Journal of Agricultural and Food Chemistry, 58(24), 12664-12671.
Ok, H. E., Choi, S.-W., Kim, M., & Chun, H. S. (2014). HPLC and UPLC methods for the
SC
determination of zearalenone in noodles, cereal snacks and infant formula. Food Chemistry, 163, 252-257.
Pei, R., Cheng, Z., Wang, E., & Yang, X. (2001). Amplification of antigen–antibody interactions based on biotin labeled protein–streptavidin network complex using impedance spectroscopy.
M AN U
Biosensors and Bioelectronics, 16(6), 355-361.
Pei, S.-C., Lee, W.-J., Zhang, G.-P., Hu, X.-F., Eremin, S. A., & Zhang, L.-J. (2013). Development of anti-zearalenone monoclonal antibody and detection of zearalenone in corn products from China by ELISA. Food Control, 31(1), 65-70.
Sai, N., Chen, Y., Liu, N., Yu, G., Su, P., Feng, Y., Zhou, Z., Liu, X., Zhou, H., Gao, Z., & Ning, B. a. (2010). A sensitive immunoassay based on direct hapten coated format and biotin–streptavidin system for the detection of chloramphenicol. Talanta, 82(4), 1113-1121.
TE D
Sheng, Y., Jiang, W., De Saeger, S., Shen, J., Zhang, S., & Wang, Z. (2012). Development of a sensitive enzyme-linked immunosorbent assay for the detection of fumonisin B1 in maize. Toxicon, 60(7), 1245-1250.
Tian, J., Zhou, L., Zhao, Y., Wang, Y., Peng, Y., & Zhao, S. (2012). Multiplexed detection of tumor markers with multicolor quantum dots based on fluorescence polarization immunoassay.
EP
Talanta, 92, 72-77.
Trapiella-Alfonso, L., Costa-Fernández, J. M., Pereiro, R., & Sanz-Medel, A. (2011). Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk. Biosensors and Bioelectronics, 26(12), 4753-4759.
AC C
448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491
Välimaa, A.-L., Kivistö, A. T., Leskinen, P. I., & Karp, M. T. (2010). A novel biosensor for the detection of zearalenone family mycotoxins in milk. Journal of microbiological methods, 80(1), 44-48.
Vinayaka, A. C., & Thakur, M. S. (2010). Focus on quantum dots as potential fluorescent probes for monitoring food toxicants and foodborne pathogens. Analytical and Bioanalytical Chemistry, 397(4), 1445-1455. Wang, L., Zhang, L.-J., Lv, W., Han, S.-H., Zhang, F.-K., & Pan, J.-R. (2011). Determination of Organophosphorus Pesticides Based on Biotin-Avidin Enzyme-Linked Immunosorbent Assay. Chinese Journal of Analytical Chemistry, 39(3), 346-350. Wang, L., Zhang, Y., Gao, X., Duan, Z., & Wang, S. (2010). Determination of Chloramphenicol Residues
in
Milk
by
Enzyme-Linked
Immunosorbent
Assay:
Improvement
by
Biotin−Streptavidin-Amplified System. Journal of Agricultural and Food Chemistry, 58(6),
16
ACCEPTED MANUSCRIPT 3265-3270. Wang, Y.-K., Yan, Y.-X., Mao, Z.-W., Wang, H.-a., Zou, Q., Hao, Q.-W., Ji, W.-H., & Sun, J.-H. (2013). Highly sensitive electrochemical immunoassay for zearalenone in grain and grain-based food. Microchimica Acta, 180(3-4), 187-193. Wang, Y., Liu, N., Ning, B., Liu, M., Lv, Z., Sun, Z., Peng, Y., Chen, C., Li, J., & Gao, Z. (2012). Simultaneous and rapid detection of six different mycotoxins using an immunochip. Biosensors and Bioelectronics, 34(1), 44-50.
RI PT
Xu, W., Xiong, Y., Lai, W., Xu, Y., Li, C., & Xie, M. (2014). A homogeneous immunosensor for AFB1 detection based on FRET between different-sized quantum dots. Biosensors and Bioelectronics, 56, 144-150.
Yang, A., Zheng, Y., Long, C., Chen, H., Liu, B., Li, X., Yuan, J., & Cheng, F. (2014). Fluorescent immunosorbent assay for the detection of alpha lactalbumin in dairy products with monoclonal antibody bioconjugated with CdSe/ZnS quantum dots. Food Chemistry, 150,
SC
492 493 494 495 496 497 498 499 500 501 502 503 504 505
73-79.
Figures Captions
507
Fig 1. Standard curves of improved icELISA, BA-ELISA and BA-FLISA for ZEN
508
determination in PBS buffer. X axle presents logarithm of ZEN concentrations, and Y
509
axle represents the percentages of B/B0 and F/F0 (the ratios of absorbance and
510
fluorescence intensity in the presence and absence of ZEN) for icELISA/BA-ELISA
511
and BA-FLISA respectively. Each point presents the mean of three replicates, and
512
error bars represent the standard deviation.
513
Fig 2. Standard curves for detection of ZEN ( ), ZAN (▼), α-ZOL (●), β-ZOL (▲),
514
α-ZAL ( ), and β-ZAL (■) in BA-ELISA, which were constructed by detecting a
515
serial of standard solutions ranging from 0.01 to 10 ng mL-1. Each point represents the
516
mean of three replicates.
AC C
EP
TE D
M AN U
506
17
ACCEPTED MANUSCRIPT Table 1. IC50 and cross-reactivities (CRs) of the ZEN-related compounds in BA-ELISA Componds
Molecular structure
CRs (%)
0.18
100
RI PT
ZEN
IC50 (ng mL-1)
0.30
59.51
M AN U
SC
ZAN
AC C
α-ZOL
EP
β-ZAL
TE D
α-ZAL
β-ZOL
0.30
60.50
0.73
24.68
0.39
46.74
0.46
39.22
ACCEPTED MANUSCRIPT
corn based infant food
60 15 2 20 10 2
BA-FLISA
Found (ng mL-1)
Recovery (%)
Inter-RSD (%)
Intra-RSD (%)
Found (ng mL-1)
Recovery (%)
Inter-RSD (%)
Intra-RSD (%)
63.16±3.61 17.30±1.19 1.82±0.05 22.24±1.26 8.98±0.85 1.53±0.02
105.27 115.35 90.92 111.22 89.82 76.67
5.71 6.88 2.53 5.65 9.51 1.21
7.99 15.21 13.55 9.78 11.69 15.60
54.25±1.43 16.93±1.28 n/d 19.00±1.74 8.94±0.73 n/d
90.42 112.89 — 94.99 89.41 —
2.64 7.57 — 9.15 8.13 —
12.06 10.17 — 8.23 13.11 —
AC C
EP
TE D
n/d = not detected.
SC
Corn flour
BA-ELISA
M AN U
Matrixes
Spiked Con. (ng mL-1)
RI PT
Table 2. Analysis of ZEN-spiked corn flour and corn based baby food by BA-ELISA and BA-FLISA (n=4)
ACCEPTED MANUSCRIPT Table 3. Comparison of the analysis results for ZEN contamination in natural samples by the developed BA-ELISA, BA-FLISA and LC-MS/MS (n=4)
ZEN
ZEN
LC-MS/MS ZEN
AC C
α-ZOL -1
β-ZOL
(ng mL )
(ng mL )
(ng mL )
(ng mL )
(ng mL-1)
8.40 156.58 14.38 2.20 21.72 29.89 344.78 7.07 19.42 77.04 12.85 11.89 53.13 P (0.70) 4.19 n/d 4.80 1.57 4.12 2.78 1.03 1.60 3.54 0.99 P (0.79) n/d
n/d 165.89 13.67 n/d 9.45 10.48 202.73 P (4.31) 14.25 49.45 13.73 9.44 54.74 n/d P (4.86) n/d P (5.00) n/d P (3.73) P (3.18) n/d n/d P (4.24) n/d n/d n/d
2.39 130.55 9.08 1.53 8.20 15.13 409.72 7.13 23.69 53.58 11.69 11.99 56.04 0.42 3.50 n/d 3.36 0.95 6.38 2.04 1.09 1.85 3.68 0.31 0.59 n/d
n/d 1.07 n/d n/d n/d n/d 1.10 n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d
n/d 4.17 n/d n/d n/d n/d 3.62 0.29 n/d 0.51 0.21 n/d 0.24 n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d
P=possible concentration of existed ZEN; n/d = not detected.
-1
RI PT
-1
SC
-1
EP
Corn based baby food
BA-FLISA
M AN U
Corn flour
1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13
BA-ELISA
TE D
Matrices
Sample Numbers
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights Two BA-immunoassays were developed basing on a group specific MAb and BSAS.
Several assay parameters optimizing were performed.
RI PT
The developed immunoassays were sensitive for family ZENs determination.
Matrix effects of corn flour and corn based baby food were studied.
Validation experiments in spiking and actual samples were performed by the
AC C
EP
TE D
M AN U
SC
developed BA-immunoassays.
ACCEPTED MANUSCRIPT
Appendices for Ultrasensitive immunoassays based on biotin-streptavidin amplified system for quantitative determination of family
Na Liu
b,c
c
c
, Dongxia Nie , Zhiyong Zhao , Xianjun Meng b, a,*
SC
Aibo Wu a
RI PT
zearalenones
Key Laboratory of Food Safety Research, Institute for Nutritional Sciences,
M AN U
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, P. R. China; Key Laboratory of Food Safety Risk Assessment, Ministry of Health, Beijing, 100021, P.R. China b
College of Food Science, Shenyang Agriculture University, 120 Dongling Road,
Shenyang 110161, Liaoning, P.R. China
Laboratory of Quality & Safety Risk Assessment for Agro-products (Shanghai),
TE D
c
Ministry of Agriculture, Institute for Agri-food Standards and Testing Technology, Shanghai Academy of Agriculture Science, 1000 Jinqi Road, Shanghai 201403, P.R.
EP
China
AC C
* Correspondences to Aibo Wu, Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy
of
Sciences,
Shanghai
200031,
Republic
of
+86-21-54920296; Fax: +86-21-54922000; E-mail: [email protected]
China.
Phone:
ACCEPTED MANUSCRIPT A TABLE OF CONTENTS Fig. A.1 The operation schematics and principle of three immunoassays involved in this study. (a) icELISA, (b) BA-ELISA, (c) BA-FLISA.
RI PT
Fig. A.2 Parameters influences on the analytical characteristics of ZEN competitive standard curves in BA-ELISA. (●) represents Amax value in the absence of ZEN and (■) represents IC50 value. (a) Coating Ag and biotinylated MAb concentrations influences
SC
(b) pH influences (c) Ionic concentrations influences (d) Solvents influences. Data were obtained from standard curves performed in triplicate.
M AN U
Fig. A.3
Parameters optimization in the assay of BA-FLISA (a) Effect of coating Ag concentrations. (b) Effect of biotinylated MAb dilutions. (c) Effect of SA-QD dilutions. Each column presents the mean of three replicates, and error bars represent the standard deviation.
TE D
Table A.1
The optimal MS/MS parameters for ZENs determination. Table A.2
AC C
EP
Parameters of matrix-matched standard curves for ZEN determination
AC C
EP
TE D
M AN U
Fig. A.1
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Fig. A.2
AC C
EP
TE D
M AN U
SC
Fig. A.3
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Table A.1. The optimal MS/MS parameters for ZENs determination
ZEN
317.25
α-ZOL
319.3
β-ZOL
319.3
α-ZAL
321.3
β-ZAL
321.3
ZAN
319.3
product ions m/z
collision energy eV
131.3*
31
175.45 275.45* 130.28 160.3* 130.2 277.35* 303.35 277.45* 303.35 275.4* 205.35
25 30 38 30 34 22 22 22 22 20 24
RI PT
precursor ions m/z
SC
Mycotoxins
AC C
EP
TE D
M AN U
* quantifier ion
ACCEPTED MANUSCRIPT Table A.2. Parameters of matrix-matched standard curves for ZEN determination Formats
Working ranges (ng mL-1)
IC50 (ng mL-1)
LOD (ng mL-1)
BA-ELISA BA-FLISA BA-ELISA BA-FLISA
2.00–69.20 9.18–141.42 0.98–31.73 6.63–110.84
10.86 32.77 5.67 27.86
0.83 3.86 0.41 2.79
Matrixes Corn flour
AC C
EP
TE D
M AN U
SC
RI PT
Corn based baby food