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:

hongtao@scau.edu.cn, lixiangmei12@163.com

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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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