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Supporting material
A smartphone-based dual detection mode device integrated with
two lateral flow immunoassays for multiplex mycotoxins in
cereals
Zhiwei Liu1, Qicheng Hua1, Jin Wang1, Zaoqing Liang2, Jiahao Li2, Jinxiao Wu3, Xing
Shen1, Hongtao Lei1,*, Xiangmei Li1,*
1 Guangdong Provincial Key Laboratory of Food Quality and Safety, College of
Food Science, South China Agricultural University, Guangzhou 510642, China
2 College of Mathematics and Infromatics, College of Software Engineering, South
China Agricultural University, Guangzhou 510642, China
3 Shanxi Institute of Feed and Veterinary Drug control, Taiyuan 030000, China
* Corresponding author. Phone: +86 20 8528 3925. Fax: +86 20 8528 0270. E-mail:
[email protected], [email protected]
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Synthesis of GNPs and GNPs-Ab probes
200 mL of double-distilled water was heated to boiling under constant stirring,
then 8.0 mL of 1 % chloroauric acid solution (w/v) was added, when the solution was
boiling again, 9.25 mL of 1 % trisodium citrate (w/v) was immediately fast added.
The reaction was boiled for another 10 min, and the solution was then cooled and
reconstituted to the initial volume by adding double-distilled water, and then stored at
4 °C for further use.
GNPs signal probes for FB1, ZEN, T-2, DON and AFB1 are prepared by
electrostatic adsorption. The optimal labeling pH and the Ab amount were adjusted by
checkerboard titration. The working conditions of the key reagents in the GNPs signal
probe labeling process of five mycotoxins were shown in Table S1. With constant
gentle stirring, a certain volume of 0.2 M K2CO3 (w/v) was added to 1 mL of GNPs
solution, a certain amount of Ab was dissolved in 100 µL of 0.5% BSA solution, then
added to the pH-adjusted GNPs solution. After 15 min of reaction at room
temperature, 20 μL of 20% BSA (w/v) was added for another 15 min blocking
reaction. The reaction solution was centrifuged at 14,000 g for 10 min at 4 °C, the
supernatant was discarded and the bottom red precipitate was dissolved with 200 μL
of resuspension. The resuspension was stored at 4 °C for further use.
Preparation of TRFMs immunolabeled Ab probes
The active ester method is used to activate the carboxyl groups on the surface of
the TRFMs and then covalently coupled with the Ab. The working conditions of the
key reagents in the TRFMs signal probe labeling process of five mycotoxins were
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shown in Table S2. With gentle stirring, 0.1 mg of TRFMs was added in 1 mL of
MES buffer (50 mM), 15 μL of freshly prepared EDC solution (0.5 mg/mL) and 20
μL of NHS solution (0.5 mg/mL) were sequentially added to the above solution. After
activation for 15 min, then centrifuged at 14,000 g for 10 min at 4 °C. The
precipitate was resuspended with 1 mL of boric acid buffer (BB, 50 mM, pH 8.0). A
certain amount of Ab was dissolved in 100 µL of BB (0.002 M, pH 8.0), then added
to the resuspension and mixed thoroughly, after incubation for another 30 min, 20 μL
of 20% BSA (w/v) was added dropwise for 15 min of blocking reaction. The mixture
was then centrifuged at 14,000 g for 10 min at 4 °C, the precipitate at the bottom of
the tube was dissolved with 200 μL of resuspension, which was the target TRFMs
immunoconjugates.
LC-MS/MS analysis
For the analysis of naturally contaminated samples, a Shimadzu (Nexera x2,
Japan) LC system and an AB Sciex triple quadrupole EMR (QTRAP®4500, USA)
were used, equipped with Analyst software for data processing. An Agilent C18
column (InfinityLab Poroshell 120 EC-C18, 4.6×150mm 2.7-Micron) was used for
chromatographic separation, and the column was kept at 30 . The sample injection℃
volume was 10 μL. The mobile phase was consisted of 0.1% acetic acid (mobile
phase A) and 100% methanol (mobile phase B), and the mobile phase flow rate was
700 μL/min. The gradient elution procedure was carried as follow: 0~5min, 5%~90%
B; 5~7min, 90% B; 7~7.1min, 90%~5% B; 7.1-10min, 5% B.
Mass analyses were performed in multiple reaction monitoring (MRM) mode
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and carried out by electrospray ionization (ESI) sources in positive-ion mode. The
spray voltage was 5.5 kV. Capillary temperature was set at 550 °C. Curtain gas, spray
gas and auxiliary gas maintained at pressure of 30, 50 and 50 psi, respectively.
The pretreatment method of the samples was as follows: Briefly, the ground sample
(10 g) was extracted with 40 mL of extracting solution (acetonitrile: water: acetic
acid, 79:20:1) and vortexed for 60 min on an orbital shaker. After centrifugation at
5000 g for 10 min, the sample extract was purified using 0.5 mL of the final extract
diluted with the same amount of acetonitrile: water: acetic acid (20: 79: 1) and then a
second purification was conducted using a syringe filter (0.22 μm).
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Figures/Tables captions
Fig. S1 The color intensity and inhibition results of A) the amount of K2CO3; B) the
amount of Abs; C) Abs dilution buffer for the GNPs-LFIA; D) the activated pH value;
E) the amount of Abs; F) Abs dilution buffer for the TRFMs-LFIA
Fig. S2 Qualitative test results of FB1/ZEN/T-2/DON/AFB1 in cereals. A) the vLODs
were 10/2.5/1.0/10/0.5 μg/kg for the GNPs-LFIA, respectively; B) the vLODs were
2.5/0.5/0.5/2.5/0.5 μg/kg for the TRFMs-LFIA, respectively. Standard curves for
quantitative detection of FB1/ZEN/T-2/DON/AFB1 in cereals. C) GNPs-LFIA; D)
TRFMs-LFIA
Fig. S3 Characterization of label materials, transmission electron microscopy images
of A) GNPs; B) TRFMs
Table S1 Working conditions of the GNPs-LFIA
Table S2 Working conditions of the TRFMs-LFIA
Table S3 Analytical performances of GNPs-LFIA and TRFMs-LFIA
Table S4 The IC50 and CR values of each mAb determined by icELISA, GNPs-LFIA
and TRFMs-LFIA
Table S5 Determination of FB1, ZEN, T-2, DON, and AFB1 in naturally
contaminated samples by LC-MS/MS, GNPs-LFIA and TRFMs-LFIA (n=3)
Table S6 Comparison of the GNPs-LFIA and TRFMs-LFIA
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Fig. S1 The color intensity and inhibition results of A) the amount of K2CO3; B) the amount of Abs; C) Abs dilution buffer for the GNPs-LFIA;
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D) the activated pH value; E) the amount of Abs; F) Abs dilution buffer for the TRFMs-LFIA93
Fig. S2 Qualitative test results of FB1/ZEN/T-2/DON/AFB1 in cereals. A) the vLODs
were 10/2.5/1.0/10/0.5 μg/kg for the GNPs-LFIA, respectively; B) the vLODs were
2.5/0.5/0.5/2.5/0.5 μg/kg for the TRFMs-LFIA, respectively. Standard curves for
quantitative detection of FB1/ZEN/T-2/DON/AFB1 in cereals. C) GNPs-LFIA; D)
TRFMs-LFIA
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Fig. S3 Characterization of label materials, transmission electron microscopy images
of A) GNPs; B) TRFMs
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Table S1 Working conditions of the GNPs-LFIA
Working conditions AFB1 ZEN DON T-2 FB1
Particle size and color 40 nm, red
Ag AFB1-
BSA
ZEN-
OVA
DON-
BSA
T-2-BSA FB1-
BSA
Ag concentration
(mg/mL)
5 6.1 6 2.8 4.1
Amount of Ag
(mg/cm)
1.3×10-4 1.6×10-4 3.2×10-4 7.5×10-5 3.3×10-4
Amount of goat anti-
mouse IgG (mg/cm)
2.8×10-4
Coating buffer 0.02 M PB (pH 7.4, 0.15 M NaCl)
The optimal labeling
pH (0.2 M k2CO3
amount)
pH 7.1
(8 μL)
pH 7.1
(8 μL)
pH 7.6
(10 μL)
pH 7.4
(9 μL)
pH 10.7
(30 μL)
Ab concentration
(mg/mL)
1.0 5.0 3.7 2.4 5.3
Amount of Ab
labeling (mg)
1.0×10-3 2.5×10-3 2.8×10-3 2.4×10-3 5.3×10-3
Coupling ratio (mass
ratio)
1:0.0025 1:0.0063 1: 0.007 1:0.006 1:0.013
Volume of
immunoprobe
2 μL 6 μL 4 μL 1 μL 2.5 μL
Ab dilution buffer 0.5%BSA
Ab probe resuspension 0.02 M PB (pH 7.4, 0.5% BSA, 0.5% tween-20, 5 %
sucrose, 0.2 % PVP, and 0.03% procline-300)
Sample pad material RB65
sample pad
pretreatment solution
0.05 M PB (pH 7.4, 0.5 % Tween-20, 0.3 % PVP, and 5 %
sucrose)
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Table S2 Working conditions of the TRFMs-LFIA
Working conditions AFB1 ZEN DON T-2 FB1
Particle size and color 200 nm, red
Amount of Ag
(mg/cm)
8×10-5 4.9×10-4 4.8×10-4 4.5×10-4 6.6×10-4
Amount of goat anti-
mouse IgG (mg/cm)
2.3×10-4
Coating buffer 0.02 M PB (pH 7.4, 0.15 M NaCl)
Activated pH value
(0.05 M MES)
pH 6.5 pH 6.5 pH 6.0 pH 5.0 pH 6.5
Coupling buffer 0.05 M BB (pH 8.0)
Amount of Ab labeling
(mg)
7.5×10-4 3.8×10-3 2.8×10-3 7.2×10-3 8.0×10-3
Coupling ratio (mass
ratio)
1:0.0075 1:0.038 1:0.028 1:0.072 1:0.08
Volume of
immunoprobe
1 μL 7 μL 4 μL 2.5 μL 2 μL
Ab dilution buffer 0.002 M BB (pH 8.0)
Ab probe resuspension 0.02 M PB (pH 7.4, 0.5% BSA, 0.5% Tween-20, 5 %
trehalose, 0.2 % PVP, and 0.03% procline-300)
Sample pad material RB65
sample pad
pretreatment solution
0.05 M PB (pH 7.4, 0.5 % Tween-20, 0.3 % PVP, and 5
% sucrose)
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Table S3 Analytical performances of GNPs-LFIA and TRFMs-LFIA
MycotoxinsvLODa
(μg/kg)qLODb
(μg/kg)IC50
c
(μg/kg )Dynamic ranged (μg/kg)
GNPs-LFIA
FB1 10 0.59 2.81 0.5-10
ZEN 2.5 0.24 1.43 0.25-5
T-2 1.0 0.32 0.63 0.3-1
DON 10 0.90 6.09 1-20
AFB1 0.5 0.27 0.36 0.25-0.5
TRFMs-LFIA
FB1 2.5 0.42 1.00 0.25-5
ZEN 0.5 0.10 0.18 0.01-1
T-2 0.5 0.05 0.15 0.05-0.5
DON 2.5 0.75 2.76 1-10
AFB1 0.5 0.04 0.17 0.05-0.5
avLOD was defined as the minimum mycotoxin to produce colorless T lines. bqLOD was determined as the concentration that gave 80% B/B0 values from the standard curves. cDynamic range was the linearity range of the standard curves (from the lowest concentration points to the maximum concentration points). dIC50 was the half maximal inhibitory concentrations of the standard curves.
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Table S4 The IC50 and CR values of each mAb determined by icELISA, GNPs-LFIA and TRFMs-LFIA
Analytes AnaloguesicELISA GNPs-LFIA TRFMs-LFIA
IC50 (μg/L) CR% IC50 (μg/L) CR% IC50 (μg/L) CR%
FB1 - 2.06 100 2.81 100 1 100
FB2 6.65 31.0 8.03 35.0 2.38 42.0
FB3 2.17 94.9 3.16 88.9 0.98 102.0
ZEN/T-2/DON/AFB1/OCT >10000 <0.1 >10000 <0.1 >10000 <0.1
ZEN - 0.048 100 1.43 100 0.18 100
α-zearalenol 0.068 70.6 2.31 61.9 0.26 69.2
β-zearalenol 0.086 55.8 2.98 48.0 0.35 51.4
α-zearalanol 0.15 32.0 5.72 25.0 0.52 34.6
β-zearalanol 0.19 25.3 7.95 18.0 0.63 28.6
zearalanone 0.21 22.9 8.03 17.8 0.67 26.9
FB1/T-2/DON/AFB1/OCT >10000 <0.1 >10000 <0.1 >10000 <0.1
T-2 - 0.39 100 0.63 100 0.15 100
HT-2 1.12 34.8 2.34 26.9 0.42 35.7
ZEN/FB1/DON/AFB1/
OCT>10000 <0.1 >10000 <0.1 >10000 <0.1
DON - 2.88 100 6.09 100 2.76 100
3-AC-DON 0.83 347 2.17 280.6 0.61 452.5
15-AC-DON 8.46 34.0 23.55 25.9 7.12 38.8
NIV 57.23 5.0 209.32 2.9 33.06 8.3
ZEN/T-2/FB1/AFB1/OCT >10000 <0.1 >10000 <0.1 >10000 <0.1
AFB1 - 0.018 100 0.36 100 0.17 100
AFB2 0.073 24.7 1.71 21.1 0.56 30.4
AFG1 0.12 15.0 2.35 15.3 1.02 16.7
AFG2 0.36 5.0 10.98 3.3 2.38 7.1
AFM1 0.04 45.0 0.86 41.9 0.35 48.6
ZEN/T-2/DON/FB1/OCT >10000 <0.1 >10000 <0.1 >10000 <0.1
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Table S5 Determination of FB1, ZEN, T-2, DON, and AFB1 in naturally
contaminated samples by LC-MS/MS, GNPs-LFIA and TRFMs-LFIA (n=3)
Samp
le
LC-MS/MS (μg/kg) GNPs-LFIA (μg/kg) TRFMs-LFIA (μg/kg)
FB1 ZEN T-2 DON AFB1FB1 ZEN T-2 DON AFB1FB1 ZEN T-2 DON AFB1
M1 11.6 5.9N
Da180.2 1 15.1 7.5 ND 207.3 1.1 13.5 5.4 ND212.7 1.0
M2 192.5 148.7 ND311.2 1 236.8 161.8 ND 276.8 1.2 225.0 153.0 ND278.6 0.8
M3 900.3 634.9 ND1269 0.7 867.7 689.4 ND 1191.4 0.6 869.6 637.8 ND1147.8 1.2
M41474.
8375.3 ND427.2 1
1522.
5 357.2 ND 456.9 1.2
1402.
1 364.7 ND439.4 0.9
M51131.
2388.7 ND543.8 1.5
1234.
8 415.6 ND 506.0 1.7
1064.
9 395.4 ND537.0 1.7
M61495.
5
1331.
3ND510.8 18.5
1284.
6
1437.
4 ND 538.1 18.6
1346.
6
1473.
1 ND521.0 18.1
M7 524.1 41.4 ND2246.3 8.7 589.8 38.6 ND 2122.1 8.6 487.7 36.0 ND2163.8 9.2
M8 720.61257.
9ND64.4 1.1 705.8
1379.
9 ND 71.2 1.3 727.3
1184.
0 ND69.1 1.5
M9 581.6 517.1 ND61.9 1.9 554.4 551.5 ND 65.5 2.1 549.0 569.2 ND73.0 2.3
M10 304.8 15.1 ND108.5 0.6 286.9 18.5 ND 95.2 0.8 347.3 16.4 ND116.9 0.7
M11 25.2 140 ND679.2 0.6 24.4 130.3 ND 708.5 0.7 26.4 144.9 ND671.1 0.8
M121398.
518.7 ND155.1 0.7
1162.
3 20.9 ND 188.1 0.8
1313.
1 16.9 ND163.7 0.9
M13 58.4 19.2 ND76 0.7 59.7 20.5 ND 65.0 1.0 55.5 19.1 ND72.6 0.5
M14 59.1 18.7 NDND 0.8 53.8 17.4 ND ND 0.8 56.5 19.7 NDND 0.8
M15 25.3 ND NDND 0.7 26.7 ND ND ND 0.9 23.2 ND NDND 0.8
M16 57 ND NDND 0.7 58.5 ND ND ND 0.7 56.4 ND NDND 0.7
W1 11.1 49.5 ND2114.6 1.3 9.5 56.8 ND 1924.5 1.6 14.0 47.6 ND1882.9 1.8
W2 6.6 ND ND1974.6 1 7.3 ND ND 1792.7 1.0 6.4 ND ND1867.2 0.9
W3 8 14.3 ND660.1 1 7.7 11.5 ND 611.4 1.4 7.6 15.7 ND646.2 1.0
W4 56.5 1105. ND37737. 1.2 53.1 1271. ND 35023. 1.6 53.0 1262. ND34505. 0.9
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2 1 9 3 1 4
W5 6.3 53.2 ND3380.5 1.6 7.9 63.4 ND 3357.8 1.7 8.0 71.0 ND3468.9 1.5
B1 1.4 19.4 ND1663 1.1 1.5 16.9 ND 1525.6 1.3 1.7 19.4 ND1468.8 0.9
B2 3.4 29.8 ND11258.
31.8 5.4 27.1 ND
10928.
2 2.0 3.3 27.3 ND9956.3 2.0
B3 2.8 22.3 ND1584 1.9 6.8 19.3 ND 1684.6 1.8 2.3 24.4 ND1575.6 2.3
B4 4.1 ND ND3925.1 2 5.1 ND ND 4105.6 2.3 4.3 ND ND3773.4 1.6
B5 11.8 18.3 ND740.6 1.5 9.1 21.6 ND 739.1 1.4 10.9 16.0 ND718.9 1.4
B6 1.4 18.8 ND1940.2 1.4 1.3 21.6 ND 1862.1 1.3 1.4 18.3 ND1985.0 1.6
B7 1 33.8 ND853.1 1.7 0.9 35.0 ND 863.0 1.7 0.8 35.7 ND826.3 2.3
B8 7.3 59.9 ND1975.1 1.9 6.9 52.7 ND 1853.8 1.8 7.5 56.2 ND1812.2 1.9
B9 1.1 42.7 ND2456.5 1.4 1.2 43.9 ND 2504.7 1.6 1.4 44.2 ND2520.0 1.6
aND, undetectable121122
Table S6 Comparison of the GNPs-LFIA and TRFMs-LFIA
Items GNPs-LFIA TRFMs-LFIA
Nanoparticle colloidal gold time-resolved fluorescent
microsphere
Ab probe preparation time 40 min 80 min
Coupling method with Ab electrostatic adsorption covalent coupling
Ag amount (mg/per strip) AFB1-BSA: 5.9×10-5
ZEN-OVA: 7.2×10-5
DON-BSA: 1.4×10-4
T-2-BSA: 3.4 ×10-5
FB1-BSA: 1.5×10-4
AFB1-BSA: 3.6×10-5
ZEN-OVA: 2.2×10-4
DON-BSA: 2.2×10-4
T-2-BSA: 2×10-4
FB1-BSA: 3×10-4
Goat anti-mouse IgG amount
(mg/per strip)
1.3×10-4 1×10-4
Ab amount (mg/per strip) anti-AFB1 mAb: 1.0×10-5
anti-ZEN mAb: 7.5×10-5
anti-DON mAb: 5.6×10-5
anti-T-2 mAb: 1.2×10-5
anti-FB1 mAb: 6.6×10-5
anti-AFB1 mAb: 3.8×10-6
anti-ZEN mAb: 1.3×10-4
anti-DON mAb: 5.6×10-5
anti-T-2 mAb: 9×10-5
anti-FB1 mAb: 8.0×10-5
vLOD (μg/kg) FB1/ZEN/T-2/DON/AFB1:
10/2.5/1.0/10/0.5, respectively
FB1/ZEN/T-2/DON/AFB1:
2.5/0.5/0.5/2.5/0.5, respectively
qLOD (μg/kg) FB1/ZEN/T-2/DON/AFB1:
0.59/0.24/0.32/0.90/0.27,
respectively
FB1/ZEN/T-2/DON/AFB1:
0.42/0.10/0.05/0.75/0.04,
respectively
Result judgement Qualitative: by visualization
Quantitative: smartphone-based
detection device
Qualitative: under UV light
Quantitative: smartphone-based
detection device
Cost price (per strip) ~0.078 USD ~0.16 USD
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