Biotin-streptavidin system-mediated ratiometric multiplex

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Food Safety and Toxicology

Biotin-streptavidin system-mediated ratiometric multiplex immunochromatographic assay for simultaneous and accurate quantification of three mycotoxins Yanna Shao, Hong Duan, Shu Zhou, Tongtong Ma, Liang Guo, Xiaolin Huang, and Yonghua Xiong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03222 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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Journal of Agricultural and Food Chemistry

Biotin-streptavidin

system-mediated

ratiometric

multiplex

immunochromatographic assay for simultaneous and accurate quantification of three mycotoxins Yanna Shao1,3#, Hong Duan1,3#, Shu Zhou1,3, Tongtong Ma1,3, Liang Guo1,3, Xiaolin Huang1,2,3* and Yonghua Xiong1,2,3* 1

State Key Laboratory of Food Science and Technology, Nanchang University,

Nanchang 330047, P. R. China; 2

School of Food Science and Technology, Nanchang University, Nanchang 330031,

P. R. China 3

Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P. R.

China;

#

These authors contributed equally to this work

*Corresponding authors: E-mail: [email protected] ( Y.H. Xiong); E-mail: [email protected]; [email protected] (X.L. Huang)

ACS Paragon Plus Environment


1

Abstract: Quantitative multiplex immunochromatographic assay (mICA) has

2

received increasing attention in multi-target detection. However, the quantitative

3

results in the reported mICAs were obtained by recording the signals on the test lines

4

that are readily interfered by various analyte-independent factors, resulting in

5

inaccurate quantitation. Ratiometric strategy using the T/C value (ratios of signals on

6

the test line to those of the control line) for signal correction can effectively

7

circumvent these issues to enable more accurate detection. Herein, we first presented

8

a novel ratiometric mICA strip with multiple T lines for the simultaneous quantitative

9

detection of aﬂatoxin B1 (AFB1), fumonisin B1 (FB1), and ochratoxin A (OTA) using

10

highly luminescent quantum dot nanobead (QB) as enhanced signal reporters. To

11

achieve reliable ratiometric signal output, a biotin-streptavidin (SA) system was

12

introduced to replace conventional anti-mouse IgG antibody for reliable reference

13

signals on the control line that are completely independent of the signal probe and

14

analyte. By using the stable T/C value as quantitative signals, our proposed QB-mICA

15

method can successfully detect three mycotoxins with concentrations as low as 1.65

16

pg/mL for AFB1, 1.58 ng/mL for FB1, and 0.059 ng/mL for OTA. The detection

17

performance of the developed QB-mICA strip, including precision, specificity, and

18

reliability, was further evaluated using artificially contaminated cereal samples.

19

Results demonstrated the improved accuracy and reliability of quantitative

20

determination compared with anti-mouse IgG antibody. Thus, this work provided a

21

promising strategy to develop a ratiometric mICA method to accurately quantify

22

multiple analytes using the biotin-SA system, opening up a new direction in


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quantitative mICA.

24

Keywords:

25

immunochromatographic assay; test strip; mycotoxins

quantum

dot

nanobeads;

biotin-streptavidin


system;

multiplex


27 28

INTRODUCTION

29

Mycotoxins are a highly toxic secondary metabolite generated by filamentous

30

fungi.1,2 Numerous mycotoxigenic fungi share the same niches for the production of

31

toxic metabolites under similar conditions, usually causing the co-contamination of

32

multiple mycotoxins in one kind of food or feed.3,4 Thus, the simultaneous occurrence

33

of multi-mycotoxin contamination is challenging conventional single-target detection

34

methods and promoting the rapid development of various multi-analyte detection

35

technologies.5 Instrumental methods such as high-performance liquid chromatography

36

and liquid chromatography tandem mass spectrometry have been used as reference

37

methods for the simultaneous detection of multi-mycotoxin contamination due to their

38

sensitivity and accuracy.6 Nevertheless, these instrument-based techniques are time-

39

consuming and depend on expensive equipment, skilled technicians, and complicated

40

pretreatment procedures, which are not suitable as point-of-care (POC) diagnostic

41

devices for mycotoxin field application.7

42

Immunochromatographic assays (ICAs) have become one of the most

43

predominant POC diagnostic tools in the past years because of their simplicity,

44

rapidity, robustness, and low cost.8-11 In particular, multiplex ICA (mICA)-based

45

diagnostic platforms have attracted increasing attention in the field of disease control,

46

food safety, and environmental monitoring because they can provide more accurate

47

sample information, higher assay efficiency, and lower test cost compared with

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conventional single ICA.12-14 Recently, increasing attempts have been devoted to


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fabricating various mICA methods for rapidly and simultaneously monitoring the co-

50

contamination of multiple mycotoxins in agro-food.15-17 However, most of these

51

reported mICAs focused on the qualitative evaluation of multi-mycotoxin residues on

52

the basis of the visual appearance of multiple test lines, which may lead to inaccurate

53

detection results due to subjective differences among individuals.18-20 Compared with

54

qualitative analysis, the quantitative measurement in ICA is relatively objective

55

because signal readout relies on the strip readers. For example, several quantitative

56

mICAs have been reported for the simultaneous determination of multiple mycotoxins

57

by recording the signal fluctuations on the test lines. Nonetheless, the quantitative

58

results are unreliable as the batch variance of strips, immunoreaction time, and sample

59

matrix can affect the capture efficiency of signal probes at the test lines, which causes

60

nonspecific and misleading signal variations, producing false negative or positive test

61

results.21

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Previous studies demonstrated that ratiometric signal output using the ratio of the

63

detection signal of the test (T) zone to that of the control (C) zone (denoted as T/C)

64

can effectively eliminate the inherent heterogeneity of ICA strips and the interference

65

from the sample matrix, thus allowing for accurate and reliable quantification of

66

target analytes.22-25 Numerous research groups, including our group, have provided

67

multifarious ratiometric single ICA test strips for accurately quantifying various

68

analytes, including proteins, small molecules, viruses, and bacteria.26-29 However, to

69

our best knowledge, the ratiometric strategy based on the T/C ratio has not yet been

70

applied to improve the quantification accuracy of mICAs. The possible reason is that



71

the signal intensity on the C line changes not only with the concentration of the probe

72

but also with the concentration of analytes because the signals at the C line are

73

commonly produced via binding of signal probes and anti-mouse IgG antibody pre-

74

immobilized onto the NC membrane. Thus, when multiple analytes are

75

simultaneously detected in single mICA strip, the anti-mouse IgG antibody on the C

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line can bind with all signal probes against different analytes to form the signal. As a

77

result, the signal at the C line often alters with the concentration change of each signal

78

probe, thereby inducing mutual interference between analytes, especially in the

79

presence of two or more analytes. Such interference between analytes makes the

80

signal on the C zone unsuitable as a reference signal to enable reliable ratiometric

81

detection. Therefore, in the mICA, the synchronous and accurate qualification for

82

multiple analytes still remains a huge challenge.

83

An appropriate C line signal system independent of the concentrations of the

84

analyte and signal probe should be promoted and developed to obtain an accurate T/C

85

ratio for achieving ratiometric measurement in mICA.30-32 To this end, the biotin-

86

streptavidin (SA) system was introduced in this study as a reliable signal output on the

87

C line, in which the signal change is only related to the sample matrix and the

88

inherent heterogeneity of the test strips, but not the analyte and probe

89

concentrations.33 Aflatoxin B1 (AFB1), fumonisin B1 (FB1), and ochratoxin A (OTA)

90

are three common mycotoxins that frequently co-occur in contaminated cereal

91

samples.34-37 To reduce their threats to human and animal health, a novel quantum dot

92

nanobead (QB)-based mICA (QB-mICA) with three T lines (T1, T2, and T3) and one


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independent C line was constructed for the simultaneous and accurate quantitation of

94

these three mycotoxins using ratiometric signal output (Scheme 1).38 Under the

95

developed conditions, further evaluation of the quantitative performance of the QB-

96

mICA, including the limit of detection (LOD), half-maximal inhibitory concentration

97

(IC50), linear detection range, accuracy and precision, and reliability, was

98

implemented in the PB solution and artificially contaminated cereal samples. Briefly,

99

this work provides a promising strategy to develop ratiometric mICA for rapidly and

100

accurately screening multi-mycotoxins in cereals, and the approach can be readily

101

extended to monitor the simultaneous concurrence of other types of analytes.39,40



103

MATERIALS AND METHODS

104

Materials and reagents. Highly luminescent QBs with maximum emission

105

wavelength of 618 nm were obtained according to our previous work.26 Bovine serum

106

albumin (BSA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), SA and

107

sodium dodecyl sulfonate were purchased from Sigma-Aldrich Chemical (St. Louis,

108

MO). AFB1-BSA (mole ratio of 20:1), FB1-BSA and OTA-BSA (mole ratio of 10:1),

109

biotin-BSA (mole ratio of 15:1), anti-AFB1 monoclonal antibodies (anti-AFB1 mAbs),

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anti-FB1 mAbs, and anti-OTA mAbs were synthesized in our lab. The sample pad,

111

absorbent pad, and NC membrane were provided by Wuxi Zodolabs Biotech Co., Ltd.

112

(Jiangsu, China). All other chemicals of analytical grade were purchased from

113

Sinopharm Chemical Corp. (Shanghai, China).

114

Synthesis of QBs-mAbs probes and QBs-SA conjugates. Anti-AFB1 (FB1,

115

OTA) mAbs and SA were conjugated with QBs via covalent coupling after

116

electrostatic adsorption following a previous report with slight modification.41 Briefly,

117

37.5 μg of unpurified ascetics or 37.5 μg of SA was added dropwise into 1 mL of PB

118

solution (0.01 M, pH 6.0) containing 250 μg of QBs, and the resultant mixture was

119

incubated under magnetic stirring at room temperature. After 45 min, 2 μL of EDC (1

120

mg/mL) was slowly added into the above mixture. After 90 min of reaction, the as-

121

prepared QBs-mAbs and QBs-SA were collected via centrifugation at 14000 × g for

122

15 min. Finally, four QB conjugates with concentration of 0.25 mg/mL, including

123

QBs-AFB1, QBs-FB1, QBs-OTA, and QBs-SA, were re-suspended in 1 mL of PBS

124

(0.01 M, pH 7.4) containing 2% fructose, 1% PEG20000, 5% sucrose, 1% BSA, and


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0.4% Tween-20. Fabrication of the QB-mICA strip. Similar to conventional single ICA strip,

127

the construction of our designed QB-mICA strip also includes the following four parts:

128

sample pad, NC membrane, absorbent pad, and backing card. The sample pad was

129

first treated with a 0.01 M pH 7.0 PBS solution containing 0.5% (v/v) Tween-20,

130

0.5% BSA, and 0.02% NaN3 followed by drying at 60 ℃ for 2 h. To obtain the

131

detection area, AFB1-BSA (1 mg/mL), FB1-BSA (1 mg/mL), OTA-BSA (1 mg/mL),

132

and biotin-BSA (2 mg/mL) or anti-mouse IgG antibody (1 mg/mL) were sprayed onto

133

the NC membrane as three T lines (T1 for AFB1 detection, T2 for FB1 detection, and

134

T3 for OTA detection) and one C lines at the dispensing rate of 0.40 μL/cm. The

135

distance between adjacent lines was set at a 4.0 mm interval. The QB-mICA strip was

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assembled by laminating the sample pad, NC membrane, and absorbent pad onto a

137

backing card and then divided into 4 mm-wide strips.

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Simultaneous quantitative detection of AFB1, FB1, and OTA using QB-

139

mICA. The quantitative detection of our developed QB-mICA strip was conducted by

140

adding 6 μL of the mixed QBs-mAbs probe solutions (2 μL of QBs-AFB1, 1 μL of

141

QBs-FB1, and 2 μL of QBs-OTA with or without 1 μL of QBs-SA) into 70 μL of

142

mycotoxin standard or sample extraction solution at the desired concentrations of

143

AFB1 from 0 to 250 pg/mL, FB1 from 0 to 50 ng/mL, and OTA from 0 to 10 ng/mL.

144

The resultant mixture solutions were incubated in the microplate well for 3 min and

145

then moved into the sample well of the strip for succeeding analysis. The fluorescence

146

intensities (FIs) at four lines were recorded after 15 min by using a commercial



147

fluorescent scanning reader. The data processing was expressed as the FIT/FIC ratio on

148

the basis of the FI value on the T1, T2, or T3 line against that of the C line. The

149

concentrations of three target analytes were quantified using the corresponding

150

standard curve obtained by plotting (BX1/B01 × 100%), (BX2/B02 × 100%), or (BX3/B03

151

× 100%) against the concentration of each analyte, where BX1 (BX2 or BX3) and B01

152

(B02 or B03) are designated as the FIT/FIC values of the AFB1 (FB1 or OTA) positive

153

and negative samples, respectively.

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Assay validation with ultrahigh-performance liquid chromatography

155

(UPLC). The reliability of the proposed QB-mICA test strip was confirmed by using

156

the well-established UPLC method. Maize, rice, and wheat sample extraction and

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UPLC operation were performed according to the national standard GB/T 5009.22-

158

2016 (China) with some modifications. The detailed procedures were taken in

159

accordance with a previous method.28


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RESULTS AND DISCUSSION

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Characterization of QBs. QBs consisting of numerous QDs embedded into

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polymer nanobeads were selected as labeling probe for the fabrication of mICA strip

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because of their strong fluorescence emission and excellent chemical colloid stability.

165

Transmission electron microscopy (TEM) image in Figure 1A indicated that the

166

obtained QBs showed a compact QD-polymer structure with regular spherical shapes

167

and relatively uniform size distribution with an average diameter of 113.7 ± 12.7 nm

168

(n = 100, Figure 1B). Dynamic light scattering analysis in Figure 1C demonstrated the

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average hydrodynamic diameter of QBs at 127.9 nm with the polydispersity index of

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0.067, revealing good monodispersity. Fluorescence spectrum analysis in Figure 1D

171

showed that the maximum emission peak of QBs was centered at 617 nm, similar to

172

that of the original QDs. However, the QBs exhibited approximately 924-fold higher

173

fluorescence emission compared with QDs alone (the detailed calculation is described

174

in the Supporting Information), thus contributing to increasing the detection

175

sensitivity of mICA using the QBs as signal reporters. Figure 1E shows the decreased

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FIs of the QBs with pH value below 7, indicating the high susceptivity of QBs to

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acidic environments. Figure 1F shows that no obvious changes in the average

178

hydrodynamic diameter and FIs of the QBs were observed after 30 days of storage at

179

room temperature, suggesting excellent long-term storage stability. Therefore, the

180

high luminescence and excellent stability of QBs make them suitable as a robust

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fluorescent label for constructing highly sensitive mICA strips.

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Fabrication of the ratiometric QB-mICA. To obtain better competitive



183

inhibition rates and appropriate FIs on both T and C lines in the competitive QB-

184

mICA, the assay development was performed by optimizing the saturated labeled

185

mAbs and SA amounts on the QB surface, used amounts of the QBs-mAbs probe

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(QBs-AFB1, QBs-FB1 and QBs-OTA) and QBs-SA probe, and concentrations of

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competitive antigens (AFB1-BSA, FB1-BSA, and OTA-BSA). In accordance with the

188

experimental results, the optimal combinations were as follows: the saturated labeling

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amounts of mAbs and SA are 150 μg of proteins per mg QBs (Figure S1); the amount

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of QBs-SA for each strip is 12.5 pg to produce an appropriate and sufficient FIC value

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at ~500-600 on the C line; the amounts of QBs-mAbs probes for each strip are 0.5 μg

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for QBs-AFB1 (Table S1), 0.25 μg for QBs-FB1 (Table S2), and 0.5 μg for QBs-OTA

193

(Table S3); and the concentrations of AFB1-BSA, FB1-BSA, and OTA-BSA on the T1,

194

T2, and T3 lines are 1 mg/mL (Tables S1-3). Under the developed conditions, the

195

immunological kinetics analyses of FIT and FIT/FIC were conducted by running a

196

blank PB solution containing QBs-mAbs onto the QB-mICA test strips with two

197

modes of C line (biotin-BSA and anti-mouse IgG antibody). The corresponding

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immunodynamic curves were recorded by plotting the values of FIT and the FIT/FIC

199

values against immunoreaction time. As shown in Figures 2A and 2B, in two modes,

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all FIT values at the T1, T2, and T3 lines shared similar continuous increasing trend

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without balance during 25 min of observation time, whereas all FIT/FIC values

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reached an equilibrium state within 10 min, suggesting that the FIT/FIC values in the

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mICA strip can also serve as a ratiometric signal readout to obtain rapid and reliable

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quantitative determination of multiple analytes in both modes. The FIC changes in two


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C line modes were investigated by running the AFB1-spiked sample solutions

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containing a series of AFB1 concentrations with or without the other two analytes.

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Results in Figure 2C revealed that compared with the negative control (marked as 0

208

pg/mL of AFB1), no obvious variations in the FIC values were observed at all detected

209

AFB1 concentrations in the presence of the other two analytes. This finding indicated

210

that the biotin-SA-mediated C line system is completely independent of target

211

analytes and detection probes, which demonstrated that the biotin-SA-mediated C line

212

system can provide a constant reference signal to the fluorescence signal at the T line

213

to allow ratiometric signal output using the FIT/FIC ratio. By contrast, the FIC values

214

based on the anti-mouse IgG antibody always fluctuated with the changes of QBs-

215

mAbs concentrations when multiple analytes were present in the sample (Figure 2D),

216

which is not suitable for ratiometric measurement.

217

To better describe the feasibility of using the biotin-SA system in developing

218

reliable control signal on the C line, we employed the above two C line modes to

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fabricate two QB-mICA strips for the simultaneous quantitative detection of AFB1,

220

FB1, and OTA. The mycotoxin standard solutions in concentrations from 0 pg/mL to

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250 pg/mL for AFB1, from 0 ng/mL to 50 ng/mL for FB1, and from 0 ng/mL to 10

222

ng/mL for OTA were tested using the developed two QB-mICA strips. Figures 2E and

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2F show the linear detection results of AFB1 by plotting BX1/B01 × 100% against the

224

logarithmic concentrations of AFB1 using these two test strips in the presence of one,

225

two, and even three analytes. Figure 2E shows that when the biotin-SA system was

226

used as the control signal system on the C zone, no significant difference in the



227

quantitative determination of AFB1 was observed in the presence of different analytes,

228

and all IC50 values were almost identical with negligible changes. On the contrary, the

229

existence of the other two analytes obviously influenced the AFB1 quantification

230

given the remarkable difference in the IC50 values when the anti-mouse IgG antibody

231

was applied as C line (Figure 2F), resulting in inaccurate results. Similar linear

232

detections for FB1 and OTA were completed and summarized in Figure S2, where

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similar disturbances from the appearance of non-target analytes were also observed in

234

quantitatively monitoring FB1 and OTA using anti-mouse IgG antibody as the C line.

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Further analysis from the addition and recovery experiments exhibited that the

236

recoveries using the biotin-SA system ranged from 83.69% to 117.56%, an acceptable

237

level in rapid diagnosis system, whereas a large range of variation in the recoveries

238

from 8.80% to 151.66% was obtained using the anti-mouse IgG antibody (Table 1).

239

The possible reason is that the signal intensity at the C line based on the anti-mouse

240

IgG antibody was determined not only by the probe amount but also regulated by all

241

analytes, thereby causing significant fluctuation in the quantitative signals using

242

FIT/FIC values in QB-mICA. In comparison, the biotin-SA system that is wholly

243

independent of the detection probes and target analytes can effectively overcome the

244

above limitations to provide reliable reference signals on the C line. Moreover, the

245

above results demonstrated the great potential of using the biotin-SA system as an

246

alternative of anti-mouse IgG antibody for reliable reference C line to achieve

247

ratiometric quantitative analysis of multiple targets in mICA.

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Performance evaluation of the biotin-SA system-mediated ratiometric QB-


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mICA. Encouraged by the above results, we utilized our designed ratiometric QB-

250

mICA strip for the simultaneous and quantitative rapid screening of three mycotoxins

251

in cereal samples. Previous work demonstrated that the pH value and methanol in the

252

reaction buffer solution can influence the analytical sensitivity of the ICA strip by

253

affecting the immunoreaction efficiency.42,43 Thus, we first systematically

254

investigated the effects of these two factors. Figure 3A shows that the competitive

255

inhibition rates for AFB1-, FB1-, and OTA-positive samples (AFB1, 55 pg/mL; FB1,

256

11 ng/mL; OTA, 490 pg/mL) were first increased and then decreased with the pH

257

value ranging from 5 to 9. The maximum inhibition rates for the three targets were

258

obtained under pH 7. Meanwhile, for AFB1-, FB1-, and OTA-negative samples, all

259

three T lines possessed appropriate fluorescence signals of 450-600. Thus, the optimal

260

pH value at 7 was selected for the succeeding analysis. For mycotoxin detection, the

261

sample extract solution containing a certain amount of methanol was necessary to

262

achieve higher extraction efficiency of various mycotoxins from the contaminated

263

samples because of their strong hydrophobicity. However, high methanol content is

264

not conducive for immunoreaction. As presented in Figure 3B, the competitive

265

inhibition rate of AFB1 continuously decreased from 49.96% to 12.87% with

266

increasing methanol concentration from 0% to 40%. For FB1, the competitive

267

inhibition rate exhibited no significant changes with methanol content below 5%,

268

whereas the competitive inhibition rate obviously declined from 51.45% to 9.23%

269

with the continuous increase of methanol content. Similar phenomena were observed

270

in determining the effect of methanol on OTA detection, in which the competitive



271

inhibition rate slightly changed at the methanol concentration of less than 10%.

272

However, a sharp decrease in the competitive inhibition rate from 54.29% to 8.27%

273

was observed with further increase of methanol concentration to 40%. Given the high

274

sensitivity of our proposed QB-mICA strip for the three mycotoxins, the optimized

275

methanol amount in the reaction solution was set at 5% for subsequent experiments.

276

Under the developed experimental conditions, the competitive inhibition curves

277

for AFB1, FB1, and OTA were created by plotting the BX1/B01 × 100%, BX2/B02 ×

278

100%, or BX3/B03 × 100% values against the logarithmic concentrations of target

279

analytes, respectively. As illustrated in Figure 3C, the linear regression equations of

280

the developed ratiometric QB-mICA strip for AFB1, FB1, and OTA were described as

281

y = -21.99 ln(x) + 133.52 (R2 = 0.9838), y = -21.31 ln(x) + 99.728 (R2 = 0.989), and y

282

= -10.4 ln(x) + 41.352 (R2 = 0.9841), respectively, where y is the B/B0 value and x is

283

the target concentration. In accordance with the corresponding calibration curve, the

284

IC50 and IC10 (denoted as the competitive inhibition rate of 10% for LOD) values for

285

AFB1 were calculated as 46.94 and 1.65 pg/mL, respectively, with a dynamic

286

detection range from 0.019 pg/mL to 20 pg/mL. For FB1 detection, the IC50 and IC10

287

values were calculated as 10.31 and 1.58 ng/mL with linear detection range of 0.049

288

ng/mL to 50 ng/mL. The IC50 and IC10 values for OTA determination were calculated

289

as 0.44 and 0.059 ng/mL with a linear detection from 0.1 ng/mL to 15 ng/mL.

290

Three common cereal samples, including maize, rice, and wheat, which are often

291

concurrently contaminated by AFB1, FB1, and OTA, were used to estimate the

292

potential of our QB-mICA method for the actual detection in real samples. The


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addition and recovery trials for intra- and inter-assays were implemented by analyzing

294

the AFB1-, FB1-, and OTA-spiked sample extracts with different concentrations.

295

Figure 4A shows that all three mycotoxins exhibited high recoveries for the intra- and

296

inter-assay with a range of 83.53% to 116.97% regardless of sample types, proving

297

that our developed QB-mICA is suitable for simultaneously and accurately detecting

298

the presence of AFB1, FB1, and OTA in the complex food sample. The selectivity of

299

the QB-mICA was evaluated using structure analog of AFG1 (0.2 ng/mL) for AFB1

300

(0.2 ng/mL), FB1 (40 ng/mL), and OTA (8 ng/mL) detection, as well as three common

301

mycotoxins, including CIT, DON, and ZEN (1000 ng/mL). Figure 4B shows that even

302

high concentrations of CIT, DON, and ZEN could not induce obvious decrease of

303

FIT/FIC at three lines, whereas the distinct decreases of FIT/FIC were obtained in the

304

presence of AFB1, FB1, and OTA compared with the negative control, suggesting a

305

negligible cross reaction with them. Of note, the presence of AFG1 could cause

306

significant decline of FIT/FIC on the T1 line, indicating a certain extent of cross

307

reaction with AFG1 for AFB1 detection, which is in accordance with our previous

308

work.44 No obvious decrease in FIT1/FIC (FIT2/FIC or FIT3/FIC) was observed when

309

AFB1 (FB1- or OTA-)-positive samples were detected. By contrast, FIT2/FIC and

310

FIT3/FIC remained almost unchanged when AFB1-positive samples were detected.

311

FB1- and OTA-positive samples exhibited the same tendency. These findings

312

demonstrated the feasibility of using ICA for the simultaneous detection of AFB1, FB1,

313

and OTA without cross-reactions. A correlation analysis between the proposed QB-

314

mICA and the well-established UPLC was conducted to confirm the reliability.



315

Eighteen random cereal samples that were artificially contaminated by AFB1, FB1,

316

and OTA at different concentrations were tested simultaneously by using QB-mICA

317

and UPLC. Table 2 shows that the detection results for the three analytes from QB-

318

mICA agreed well with those obtained using UPLC, demonstrating the comparable

319

reliability of QB-mICA with UPLC in quantitatively monitoring the pollution levels

320

of AFB1, FB1, and OTA. However, the designed QB-mICA can simultaneously detect

321

all toxin-contaminated cereal samples, but at least one mycotoxin contamination was

322

not been detected in nine samples by UPLC. These results demonstrated that the

323

reported ratiometric QB-mICA platform can provide sensitive, accurate, specific, and

324

reliable quantification of multi-mycotoxin contamination simultaneously under

325

resource-limited settings.

326

In summary, here we successfully developed a novel ratiometric QB-mICA strip

327

with three T lines for the simultaneous quantitative detection of three common

328

mycotoxins (AFB1, FB1, and OTA) that usually co-occur in contaminated cereal

329

samples. The biotin-SA system, which is totally independent of the concentrations of

330

signal probe and target, was applied as a reliable signal output for the C line to

331

achieve the ratiometric signal output using the T/C value as quantitative signals.

332

Under the optimal conditions, the as-prepared QB-mICA method obtained reliable

333

linear detection for the three targets with IC50 values of 46.94 pg/mL for AFB1, 10.31

334

ng/mL for FB1, and 0.44 ng/mL for OTA and LOD values of 1.65 pg/mL for AFB1,

335

1.58 ng/mL for FB1, and 0.059 ng/mL for OTA. Moreover, the QB-mICA strip

336

demonstrated high accuracy, enhanced repeatability, and excellent selectivity. The


Page 18 of 37

Page 19 of 37


337

reliability was further verified using correlation analysis with UPLC method.

338

Collectively, this study provided a sensitive and accurate quantitative mICA tool for

339

simultaneously monitoring multiple mycotoxins in cereals, opening up a new concept

340

for designing and fabricating ratiometric mICA method to enhance quantitative

341

reliability.

342 343

Associated content

344

Supporting Information

345

Comparison of FI between CdSe/ZnS QDs and the prepared QBs, confirmation of the

346

saturation concentration of the protein conjugated with QBs, optimization of the QB

347

strip parameters.

348 349

Funding

350

This work was supported by a grant to Prof. Yonghua Xiong from the National

351

Key Research and Development Program of China (2018YFC1602203 and

352

2018YFC1602505), and Interdisciplinary Innovation Fund of Natural Science,

353

NanChang University (9166-27060003-ZD01). Dr. Xiaolin Huang was supported by

354

the Opening Fund of Jiangsu Key Laboratory for Food Quality and Safety-State Key

355

Laboratory Cultivation Base, Ministry of Science and Technology (028074911709),

356

and the Innovation Fund Designated for Graduate Students of Jiangxi Province

357

(YC2016-B012).



359

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504 505

Tables

506

Table 1 Comparison of precision and stability of the QB-mICA method in AFB1 (FB1,

507

OTA)-spiked samples in 0.01 M PB solution (pH 7.0) based on the biotin-BSA or

508

anti-mouse IgG antibody for control line. Intra-assay precision Spiked Control line Target toxin CV b Recovery (ng/mL) Mean (%) (%) Biotin-BSA AFB1 0.140 0.154 109.69 1.32 0.060 0.060 100.39 4.49 0.025 0.025 101.88 4.03 FB1 50 56.75 113.50 6.14 15 15.91 106.08 1.58 5 4.20 84.03 1.06 OTA 6.25 5.64 90.19 3.92 0.70 0.70 100.07 2.99 0.05 0.06 110.06 1.73 Anti-mouse AFB1 0.120 0.175 146.12 1.66 IgG 0.060 0.044 73.69 0.66 antibody 0.020 0.005 26.87 3.89 FB1 30 38.19 127.30 2.68 20 28.72 143.62 3.77 10 3.22 32.15 3.06 OTA 1.60 1.31 82.06 4.00 0.80 0.07 8.80 7.21 0.40 0.05 11.53 2.73

Inter-assay precisiona Recovery CV Meanb (%) (%) 0.156 111.08 2.92 0.060 100.63 1.30 0.023 93.70 7.56 58.78 117.56 3.01 16.72 111.45 7.91 4.18 83.69 2.39 5.99 95.79 5.06 0.73 104.56 8.53 0.06 113.77 2.90 0.168 139.72 6.00 0.048 80.56 7.40 0.005 23.89 11.30 36.70 122.35 4.48 30.33 151.66 7.43 3.37 33.75 8.67 1.42 88.63 11.15 0.08 9.79 11.95 0.05 11.61 7.83

509

a: Assay was completed every 1 days for 3 days continuously.

510

b: Mean value of five replicates at each spiked concentration.



512 513

Table 2 Recoveries of ICAs in AFB1, FB1 and OTA spiked maize (rice, wheat)

514

samples.

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Sample type Maize Maize Maize Maize Maize Maize Rice Rice Rice Rice Rice Rice Wheat Wheat Wheat Wheat Wheat Wheat

AFB1 18.63±2.54 8.68±0.10 1.08±0.12 0.55±0.01 0.16±0.01 0.04±0.003 22.49±3.44 9.31±0.80 1.00±0.03 0.51±0.04 0.18±0.01 0.04±0.004 21.73±1.60 11.08±1.03 1.09±0.17 0.57±0.04 0.18±0.002 0.05±0.001

strip (ng/g)a UPLC (ng/g) FB1 OTA AFB1 FB1 OTA 380.40±60.12 234.34±30.22 20.44 414.17 170.34 207.19±42.52 64.30±4.43 10.09 188.49 46.19 89.43±7.75 11.29±0.18 1.19 103.04 11.06 46.84±3.79 2.98±0.31 0.69 2.47 －b 21.66±1.55 0.48±0.015 0.43 －－ 9.92±0.63 0.04±0.001 －－－ 362.55±5.83 221.58±2.80 19.96 416.84 212.97 169.65±22.85 46.51±8.62 10.08 181.76 57.64 110.87±9.69 8.18±2.26 1.18 101.62 10.04 41.53±3.66 2.89±0.70 0.70 2.35 － 20.57±5.64 0.39±0.19 0.47 －－ 9.17±1.22 0.05±0.005 －－－ 469.84±55.19 195.48±28.62 20.61 405.29 188.43 230.63±36.85 51.50±3.09 10.26 185.88 42.14 115.67±21.75 9.56±0.91 1.19 105.29 9.35 47.31±0.76 2.92±0.25 0.70 2.35 － 20.93±2.26 0.41±0.08 0.43 －－ 11.29±0.36 0.03±0.001 －－－

515

a: Mean of three repeated determinations.

516

b: Not detected.


Page 28 of 37

Page 29 of 37


518 519

Figure Legends

520

Scheme 1. Compared with biotin-BSA and anti-mouse IgG antibody as control line

521

for the simultaneous detection of AFB1, FB1 and OTA using QB-mICA.

522

Figure 1. Characterization of the free QBs and QBs-probes. (A) TEM image of QBs

523

with different magnifications. (B) Histograms of the dimension distributions of 100

524

randomly selected QBs from TEM images in Figure 1A. (C) Hydrodynamic diameter

525

of QBs and QBs-AFB1 (FB1, OTA) probes. (D) Fluorescence spectra of CdSe/ZnS

526

QDs and the resultant QBs. The concentrations of QDs and QBs were 158 nmol/L,

527

and 171×10-3 nmol/L, respectively. (E) Fluorescence intensities of QBs water

528

dispersions under various pH values. Inset shows the photograph of QBs dispersions

529

with various pH values under UV light. (F) Hydrodynamic diameter variations and

530

Fluorescent stabilities of QBs dispersed in PBS against storage time.

531

Figure

532

situations where biotin-BSA (A) and anti-mouse IgG antibody (B) were used as

533

control lines, respectively. Effect of biotin-BSA (C) and anti-mouse IgG antibody (D)

534

as control line on FIC (where FIC of the AFB1-spiked sample solution; where FIC of

535

the AFB1 and FB1-spiked sample solution; where FIC of the AFB1 and OTA-spiked

536

sample solution; where FIC of the AFB1, FB1 and OTA-spiked sample solution. The

537

concentration of FB1 and OTA is random). Standard inhibition curve for AFB1 with

538

different control lines was obtained by plotting the normalized signal B/B0×100%

539

against the logarithm of AFB1 concentration, where B and B0 are FIT/FIC values of the

2.

Immunoreaction

dynamics

of

FIT/FIC


of

different

probe

in


540

AFB1 positive and negative samples, respectively. Data were obtained by averaging

541

three independent experiments (E) in situations where biotin-BSA was applied as

542

control line; (F) in situations where anti-mouse IgG antibody was applied as control

543

line.

544

Figure 3. (A) Effect of pH value of samples on competitive inhibition rate.

545

Competitive inhibition rate was defined as (1-B/B0)×100%, where B0 and B represent

546

FIT/FIC of the negative sample and AFB1 (FB1, OTA) -spiked sample solution (55

547

pg/mL, 11 ng/mL, 490 pg/mL). (B) Effect of methanol in samples on competitive

548

inhibition rate. (C) Optimized standard inhibition curve for AFB1, FB1 and OTA will

549

be was obtained by plotting the normalized signal B/B0×100% against the logarithm

550

of positive concentration, where B is the FIT/FIC value of AFB1, FB1, and OTA co-

551

contaminated samples, and B0 is the FIT/FIC value of three mycotoxin free samples,

552

respectively. Data were obtained by averaging three independent experiments.

553

Figure 4. (A) Precision and stability of the QB-mICA method in AFB1, FB1 or OTA-

554

spiked samples. Inter-assay precision was completed every 1 day for 3 days

555

continuously. The recovery rates of five replicates at each spiked concentration. (B)

556

Cross-reactivity of QB-mICA. Serial mycotoxin concentrations of ZEN, CIT, DON

557

and AFG1 (1000, 1000, 1000, 0.2 ng/mL for AFB1, FB1 and OTA detection; and 0.2,

558

40, 8 ng/mL of AFB1, FB1 and OTA) in 0.01 M PB solution containing 5% methanol

559

with pH 7.0. The error bars represent the standard deviation of three measurements.


Page 30 of 37

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560 561

Scheme 1



562

Figure 1


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563 564

Figure 2



565

Figure 3


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566 567

Figure 4



569 570

TOC Graphic

571 572


Page 36 of 37

Page 37 of 37


In this work, we successfully developed a novel ratiometric QB-mICA strip with three T lines for the simultaneous quantitative detection of three common mycotoxins (AFB1, FB1, and OTA) that usually cooccur in contaminated cereal samples. The biotin-SA system, which is totally independent of the concentrations of signal probe and target, was applied as a reliable signal output for the C line to achieve the ratiometric signal output using the T/C value as quantitative signals.


Biotin-streptavidin system-mediated ratiometric multiplex

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