Multiplex Lateral Flow Immunoassays Based on Amorphous Carbon

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Multiplex lateral flow immunoassays based on amorphous carbon nanoparticles for detecting three Fusantium mycotoxins in maize Xiya Zhang, Xuezhi Yu, Kai Wen, Chenglong Li, Ghulam Mujtaba Mari, Haiyang Jiang, Weimin Shi, Jianzhong Shen, and Zhanhui Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02827 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 22, 2017

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Journal of Agricultural and Food Chemistry

1

Multiplex lateral flow immunoassays based on amorphous

2

carbon nanoparticles for detecting three Fusantium

3

mycotoxins in maize

4

Xiya Zhang†, ‡, Xuezhi Yu†, Kai Wen†, Chenglong Li†, Ghulam Mujtaba Mari†,

5

Haiyang Jiang†, Weimin Shi†, Jianzhong Shen†, Zhanhui Wang†, *

6 7

†

8

of Veterinary Medicine, China Agricultural University, Beijing Key Laboratory of

9

Detection Technology for Animal-Derived Food Safety, Beijing Laboratory for Food

10

Beijing Advanced Innovation Center for Food Nutrition and Human Health, College

Quality and Safety, 100193 Beijing, People’s Republic of China

11

‡

12

Road, Zhengzhou, Henan 450002, China

College of Food Science and Technology, Henan Agricultural University, 63 Nongye

13

*

14

ZH Wang, [email protected]

15

Tel.: +86-106-273-4565; Fax: +86-106-273-1032.

Corresponding author:

1

ACS Paragon Plus Environment


16

ABSTRACT

17

The detecting labels used for lateral flow immunoassays (LFAs) have been

18

traditionally gold nanoparticles (GNPs) and more recently, luminescent nanoparticles

19

such as quantum dots (QDs). However, these labels have low sensitivity and are

20

costly, in particular for trace detection of mycotoxins in cereals. Here, we provided a

21

simple preparation procedure for amorphous carbon nanoparticles (ACNPs) and

22

described multiplex LFAs employing ACNPs as labels (ACNP-LFAs) for detecting

23

three Fusarium mycotoxins. The analytical performance of ACNPs in LFA was

24

compared with GNPs and QDs using the same immunoreagents, except for the labels,

25

allowing their analytical characteristics to be objectively compared. The visual limit

26

of detection (vLOD) for ACNP-LFAs in buffer was 8-fold better than GNPs and

27

2-fold better than QDs. Under optimized conditions, the quantitative limit of detection

28

(qLOD) of ACNP-LFAs in maize were as low as 20 µg/kg for deoxynivalenol, 13

29

µg/kg for T-2 toxin and 1 µg/kg for zearalenone. These measurements were much

30

lower than the action level of these mycotoxins in maize. The accuracy and precision

31

of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples

32

with recoveries of 84.6–109% and coefficients of variation below 13%. The results of

33

ACNP-LFAs using naturally incurred maize samples showed good agreement with

34

results from HPLC-MS/MS, indicating that ACNPs were more sensitive labels than,

35

and a promising alternative to, GNPs used in LFAs for detecting mycotoxins in

36

cereals.

2


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Keywords: amorphous carbon nanoparticles, gold nanoparticles, quantum dots,

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multiplex lateral flow immunoassay, mycotoxins

3



39

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INTRODUCTION

40

More than 400 mycotoxins with different chemical structures have been

41

identified.1 Mycotoxin contamination frequently occurs in food and animal feed and

42

has a range of adverse effects including teratogenicity, genotoxicity, immune

43

suppression and/or toxicity, and endocrine disruption, leading to global human and

44

animal health risks.2-5 The worldwide Alltech 37+® survey on more than 10,000 feed

45

samples between 2012 and 2015 for 37 mycotoxins showed that more than 97% of

46

samples were contaminated with an average of six mycotoxins per sample. More than

47

70% of samples were contaminated with deoxynivalenol (DON), a typical Fusarium

48

mycotoxin (http://www.knowmycotoxins.com/alltech-37plus). A survey by BIOMING

49

in 2015 of more than 8000 samples from 75 countries reported 84% of samples were

50

contaminated with at least one mycotoxin, DON, zearalenone (ZEN) and fumonisins

51

(FBs) were three frequently occurring toxins. DON poses the most frequent threat to

52

livestock with a prevalence of 73% and an average contamination level of 1090 µg/kg.

53

In addition, T-2 toxin (T-2) was found in 23% of samples, the highest contamination

54

concentration

55

(http://www.thefishsite.com/fishnews/27355/2015-biomin-mycotoxin-survey-out-now/).

56

Consumers around the world are paying attention to the risks associated with human

57

exposure to these mycotoxins. To protect consumers from exposure to these

58

mycotoxins, strict national standards for limits of mycotoxin levels are set by many

59

countries. To deal with the increasing number of samples, fast and accurate analytical

60

methods are urgently needed. This demand led to the development of rapid, simple

was

685

µg/kg

in

4


corn

samples

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and

easy-to-use

62

immunochemical techniques due to their sensitivity and limited detection time.

63

Considering the co-occurrence of mycotoxins in cereal grains, for efficient

64

surveillance purposes, an immunoassay that could detect multiple rather than

65

individual mycotoxins would be preferable. Recent reports on immunoassays for

66

mycotoxins

67

immunoassay formats have been employed for screening multiple mycotoxins, such

68

as fluorescent immunosorbent assays,13 flow-through immunoaffinity chromatography

69

tests,14, 15 chemiluminescent biosensors,16 suspension arrays,17 immunochip assays.18

have

screening

methods

increasingly

for

shifted

to

mycotoxin

multiple

detection

detections.6-12

based

on

Different

70

We developed a multi-wavelength fluorescence polarization immunoassay

71

(MWFPIA) for simultaneous detection of DON, T-2 and FBs.19 Although high speed

72

and throughput were achieved with MWFPIAs, the sensitivity was relatively low

73

compared to traditional enzyme-linked immunosorbent assays (ELISAs). The

74

sensitivity of FPIA may not be acceptable for analysis of trace levels of mycotoxins.

75

Limited approaches are available to enhance FPIA sensitivity, largely because it lacks

76

a signal amplification step during the detection procedure. In the last decade, lateral

77

flow immunoassays (LFAs) for detection of mycotoxins in food and feed have

78

attracted interest due to their short assay times, low interferences and costs, and easy

79

operation by non-specialized personnel.20 LFAs have the speed of FPIA without the

80

separation of reacted and unreacted compounds. They have the sensitivity of ELISA

81

with available signal amplification. Multi-LFAs have been applied in the screening of

82

multiple mycotoxins in cereals.6-12 5



83

Sensitivity and color intensity, detection capability and assay time are important

84

criteria for developing multiple mycotoxin LFAs, which are mostly dependent on the

85

performance of antibodies and labels.21 Generally, the antibodies used in LFAs are

86

highly defined and restricted with unalterable affinity and specificity. Thus, the use of

87

labels is a key for constructing sensitive LFAs. In the literature, gold nanoparticles

88

(GNPs) are the most extensively used labels for LFAs because they are easy to

89

synthesize, stable over time, and biocompatible.22-24 They have an intense red color

90

that is easily distinguished by the naked eye.25 Although LFAs with GNPs

91

(GNP-LFAs) give satisfactory performance in some cases, the application of

92

GNP-LFAs has been limited by low sensitivity and poor quantitative discrimination,

93

especially of trace level multiple mycotoxins, which are intrinsically determined by

94

the molar absorption coefficient of the GNPs.26 To increase the sensitivity of LFAs,

95

new nanomaterial labels such as quantum dots (QDs),12, 27 near-infrared fluorescence28

96

and up-converting phosphors nanoparticles29,

97

preparation procedures and high costs. Moreover, the excitation and emission

98

wavelengths of luminous labels are in the ultraviolet-visible or near-infrared region,

99

so interpretation by the naked eye in natural light conditions is difficult. The use of

100

semiconductor QDs for LFAs (QD-LFAs) is a successful application of

101

nanotechnology; however, QDs contain elements that are thought to be detrimental to

102

health and the environment.31 Thus, little improvement in performance has been

103

achieved, and alternative labels that are inexpensive and have low toxicological and

104

sensitive properties remain to be developed.

30

have been used, with complicated

6


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105

Amorphous carbon nanoparticles (ACNPs) are not typical nanomaterials. Their

106

diameter is usually more than 100 nm,32 which is different from well-developed

107

carbon nanotubes that have a single-walled or multi-walled structure. Although

108

ACNPs are not monodisperse, several studies have demonstrated that as labels in

109

LFAs, they are more sensitive than GNPs or latex beads. These results are attributed

110

to their strong dark color and high contrast against light backgrounds.33 Sensitivity in

111

the low picomolar range has been achieved using ACNPs in an LFA format, even with

112

visual inspection.34, 35 In addition, ACNPs possess other properties that make them

113

promising alternatives to GNPs such as easy preparation, high stability, absence of

114

toxicity, ease of conjugation, and lack of need for activation.36 Despite these

115

advantages, ACNPs are still relatively unknown in commercial LFA tests and the

116

literature. This may due to the limited availability of ACNPs. Also, the feasibility of

117

ACNPs in multi-LFAs has not been evaluated and ACNPs use has been applied only

118

in the detection of single pesticides, plant growth regulator and veterinary drugs in

119

food samples.21, 37-39

120

Although more than 30% of LFA papers have focused on mycotoxin detection in

121

food and feed samples,40 the application of ACNPs in LFAs (ACNP-LFAs) for

122

mycotoxin detection, especially multiple mycotoxins detection is scarce. In this paper,

123

we provided a simple procedure for preparing ACNPs and used them as labels to

124

develop LFAs for multiple mycotoxin detection. We combined previously produced

125

monoclonal antibody (mAb) 9C7 to T-2, mAb 3D4 to ZEN and a newly prepared

126

mAb to DON. The analytical performances of multiplex ACNP-LFAs were compared 7



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127

with performances of GNPs and QDs using the same immunoreagents. The

128

applicability of the multiplex ACNP-LFAs for real cereal samples was assessed using

129

HPLC-MS/MS as a reference method.

130

131

MATERIALS AND METHODS

132

Reagents and instruments

133

DON,

3-acetyl-deoxynivalenol

(3-Ac-DON),

15-acetyl-deoxynivalenol

134

(15-Ac-DON), ZEN, zearalanone, α-zearalanol, β-zearalanol, α-zearalenol

135

β-zearalenol were from Sigma-Aldrich (St. Louis, MO). Ochratoxin A (OTA) and

136

nivalenol (NIV) were from Fermentek Biotechnology (Jerusalem, Israel). T-2, HT-2

137

toxin fumonisin B1 (FB1) and aflatoxin B1 (AFB1) were from Pribolab Pte. Ltd.

138

(Singapore City, Singapore).

139

and

GNPs and polymer modified core/multishell QDs were previously prepared by

140

our group.41,

42

141

Laboratories (West Grove, PA). Bovine serum albumin (BSA) was from

142

Sigma-Aldrich. Nitrocellulose membranes (Millipore 135) were from Millipore

143

(Bedford, MA). Sample pad (CFKJ-0328) and the absorbance pad (CH37K) were

144

from Shanghai Liangxin Co. Ltd (Shanghai, China). O-carboxymethyl oxime (CMO),

145

succinic

146

hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were from Aladdin

147

Chemistry Co. Ltd (Shanghai, China). Other reagents and solvents were of analytical

Goat anti-mouse IgG was from Jackson Immuno-Research

anhydride

(HS),

1-ethyl-3-(3-dimethylaminopropyl)

8


carbodiimide

Page 9 of 41


148

grade or higher.

149

Specific mAb 9C7 against T-2 and mAb 3D4 against ZEN was previously

150

prepared.43, 44 The anti-DON mAb 12E8 was recently produced by our team and will

151

be described elsewhere. Negative and naturally contaminated maize samples were

152

kindly provided by Professor Sarah De Saeger (Ghent University) and were stored at

153

−20 °C until use.45

154

An ESE-Quant LFR fluorescence reader was from QIAGEN (Dusseldorf,

155

Germany). A ZX1000 dispensing platform and CM4000 guillotine cutting module

156

used to prepare LFAs were from BioDot, Inc. (Irvine, CA, USA). A microplate reader

157

SpectraMax M5 was from Molecular Devices (Downingtown, PA).

158

159

Preparation and characterization of the ACNPs

160

ACNPs were prepared as previously described with some modifications.46-48

161

First, smoldering candles were placed under a glass plate, and the candle soot scrap

162

off. Second, 10 mg candle soot was dispersed in 30 mL water/ethanol mixture (1:1)

163

under ultra-sonication for 4 h. The mixture was centrifuged at 3,000 g for 2 min to

164

remove large particles and supernatant was collected and centrifuged at 10,000 g for

165

10 min. Black precipitate was collected and dried at 37 °C. The obtained ACNPs

166

(about 3 mg) were characterized by transmission electron microscope (TEM),

167

dynamic light scattering (DLS), zeta potential (ZP).

168 9



169

Preparation of ACNPs-mAbs, GNPs-mAbs and QDs-mAbs

170

ACNPs-mAbs

171

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ACNPs were conjugated with mAbs through covalent cross-linking by a

172

bifunctional

glutaraldehyde

reagent

as

previously

described

with

some

173

modifications.32,33 4 mg ACNPs was suspended in 1 mL 10 mM borate buffer (pH =8),

174

and slowly added to 16 µL mAb 12E8 (25 µL mAb 9C7 or 12 µL mAb 3D4) under

175

vigorous shaking for one day at room temperature. (All three mAbs were dialyzed

176

against 10 mM borate buffer to a final concentration of 1 mg/mL). Subsequently, 50

177

µL 25% glutaraldehyde was added slowly to the carbon suspension with stirring for 2

178

h. To block unreacted sites, 100 µL 20% BSA was added and stirred for 30 min.

179

ACNPs-mAbs were obtained by centrifugation at 10,000 × g for 10 min. After

180

removing the clear supernatant, ACNPs-mAbs conjugates were dispersed and washed

181

two times with 10 mM borate buffer. ACNPs-mAbs conjugates were re-suspended in

182

1 mL 10 mM PBS containing 2% BSA and 20% glycerol, and stored at 4 °C until use.

183

ACNPs-mAbs conjugates were characterized by TEM, DLS and ZP.

184

185

186 187

GNPs-mAbs and QDs-mAbs GNPs and QDs labeled with mAbs were prepared and characterized by procedures described in Supporting Information.

188 189

Development of multi-LFAs 10


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Assembly of multi-LFAs

191

The multi-LFAs consisted of four sections: sample pad, conjugation pad, NC

192

membrane and absorbent pad. NC membranes were coated with 0.08 µL per mm

193

3-HS-DON-BSA (1.2 mg/mL), T-2-HS-BSA (2.2 mg/mL), ZEN-CMO-BSA (0.8

194

mg/mL) and goat anti-mouse antibodies (0.6 mg/mL) (Figure 1). The assembly was

195

similar to the procedure previously described by our group.41, 43 Assembled plates

196

were cut into 3 mm wide strips and stored under dry conditions at room temperature.

197

198

Detection procedure of multi-LFAs

199

The multi-LFAs system was based on competition for labeled-mAbs binding

200

sites among free mycotoxins and fixed coating antigens sprayed on NC membranes

201

(Figure 1). For example, for multiplex ACNP-LFAs, 50 µL, 70 µL and 30 µL of

202

ACNPs-mAb 12E8, ACNPs-mAb 9C7 and ACNPs-mAb 3D4 solutions were added to

203

a 1.5 mL tube, and mixtures were adjusted to 300 µL by adding 150 µL 10 mM PBS.

204

Then, 200 µL standard solution or sample solution was mixed with 30 µL mixture of

205

the three ACNPs-mAbs conjugates in a well of 96-microtiter plate and reacted at

206

room temperature for 3 min to allow antibody binding sites completely conjugate with

207

free mycotoxins. Strips were vertically inserted into micro-well for another 8 min. The

208

result was judged visually for qualitative analysis or by a strip reader for quantitative

209

analysis.

210 211

Sensitivity and specificity of multi-LFAs 11



212

Sensitivity of multi-LFAs was evaluated by analyzing a series of concentrations

213

of mycotoxin mixtures. For qualitative assays, results were obtained immediately

214

(Figure 1B). The visual limit of detection (vLOD) of assays was defined as the

215

minimum mycotoxin to produce colorless test lines. For semi-quantitative analysis,

216

the reflectance value of test lines was measured after 8 min using a hand-held strip

217

scan reader and B/B0 value (ratio of reflectance value of the test line of a positive

218

sample to value of a negative sample) was obtained (Figure 1C). The three

219

mycotoxins were quantified by a calibration curves (B/B0 versus mycotoxin

220

concentration). Quantitative limit of detection (qLOD) was determined as the

221

concentration that gave 80% B/B0 values, using calibration curves.9, 49

222

Specificity, expressed as cross-reactivity (CR%), was evaluated by assessing

223

recognition of other mycotoxins such as AFB1, FB1, NIV, HT-2, 3-Ac-DON,

224

15-Ac-DON, zearalanone, α-zearalanol, β-zearalanol, α-zearalenol and β-zearalenol.

225

CR% was expressed as percentage of the IC50 value of target analyte to analogs.

226

227

Application of multi-LFAs to maize samples

228

Assessment of matrix effects

229

Selected blank maize samples were confirmed using HPLC-MS/MS.45 Samples

230

were prepared by extracting 5.0 g maize flour with 15 mL methanol/PBS (70/30, v/v),

231

and the mixture was vigorously mixed for 3 min and centrifuged at 3,000 × g for 5

232

min. To eliminate matrix effects, supernatant was diluted 10-fold with 10 mM PBS 12


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233

for detection of ACNP-LFAs.

234

235

Accuracy and precision of multi-LFAs in maize

236

Accuracy (expressed as recovery) and precision (as coefficient of variation [CV])

237

of ACNP-LFAs were determined after spiking with mycotoxins at 40, 80, 160 µg/kg

238

for DON; 15, 30, 60 µg/kg for T-2; and 2.5, 5, 10 µg/kg for ZEN.

239

240

Comparison of multi-LFAs with HPLC-MS/MS in naturally contaminant samples

241

Naturally contaminant maize samples extracts were prepared following the

242

corresponding procedure for their analysis by multiplex ACNPs-LFA and

243

HPLC-MS/MS.45

244

245

RESULTS AND DISCUSSION

246

Characterization of coating antigens and mAbs

247

Pairs of coating antigens and mAbs are an important component that affects the

248

sensitivity and specificity of LFAs. Before constructing multi-LFAs, coating antigens

249

and mAbs were characterized to gather information on these immunoreagents about

250

molar ratios of haptens-to-BSA, affinity and specificity of antibodies in indirect 13



ELISA

(icELISA)

format.

Coating

antigens

Page 14 of 41

251

competitive

3-HS-DON-BSA,

252

T-2-HS-BSA and ZEN-CMO-BSA were identified by matrix-assisted laser desorption

253

time-of-flight mass spectrometry (MALDI-TOF-MS) (Table S1 and Figure S2).

254

Molar ratios of haptens-to-BSA were 3.4 to 19.1, implying that the coating antigens

255

were successfully prepared and able to be used for sensitive LFAs.7, 43

256

To develop multi-LFAs, a potential problem was cross-reaction (CR%) between

257

different antigen-antibody pairs. The best way to solve this problem was to strictly

258

select recognition molecules without CR%. Thus, we evaluated the CR% of three

259

immunoreagents using an icELISA format (Table S2). The mAb 12E8 showed high

260

CR% with 3-Ac-DON and 15-Ac-DON, but not recognized T-2 and ZEN. mAb 9C7

261

showed high affinity to T-2 and did not recognize HT-2. mAb 3D4 showed broad

262

specificity and uniform affinity for six ZEN analogs but not T-2 and DON. The CR%

263

of these mAbs demonstrated that they recognized only structurally similar analogs,

264

and non-specific binding of the other mycotoxins. Although an overestimation might

265

be obtained with the non-specific mAbs 12E8 and 3D4, broad-specificity could be

266

beneficial for multi-LFAs construction. The specificity of the mAbs was evaluated by

267

LFA in the following study. We successfully obtained and characterized coating

268

antigens and mAbs, and the high-performance of these immunoreagents ensured

269

development of highly sensitive multi-LFAs.

270

271

Preparation and characterization of ACNPs

14


Page 15 of 41


272

Compared to two-dimensional graphene (or graphene oxide), which has a

273

relatively large plane, the size of zero-dimensional ACNPs has better biocompatibility

274

as immunoassays labels.48 A literature survey by FIND Diagnostics showed that the

275

sensitivity of ACNPs can be in the low picomolar range, even when assays are judged

276

by visual inspection.34 However, the use of ACNPs in LFA labels is rarely reported.

277

The source of ACNPs in reported LFAs is mostly commercial named SB4, which has

278

an average size of 120 nm. The cost of the ACNPs may prevent their wide application.

279

Several studies report the preparation of ACNPs from toluene, fructose and

280

cyclodextrins by hydrothermal degradation.32,

281

preparation methods are complex, require relatively expensive starting materials, or

282

are made more expensive by a high-energy process. We developed a simple, easy and

283

inexpensive procedure for home-made ACNPs, originally developed by Pang’s group

284

with some modifications.48 The ACNPs prepared by Pang’s group were used as

285

energy acceptors in a fluorescence resonance energy transfer system; they have not

286

been used as labels in an immunoassay. The size and morphology of the obtained

287

ACNPs were studied by TEM. The obtained ACNPs were not monodisperse (Figure

288

2A), consistent with commercial ACNPs.33, 46, 49 Although this heterogeneous size

289

distribution may be a drawback, studies show it can be an advantage in most

290

applications.52 In addition, the hydrodynamic diameter was 142.4 nm by DLS (Figure

291

2E and Table S3). Compared with previously reported ACNPs, the ACNPs we

292

obtained were bigger than those derived from commercial SB4 and would be more

293

comparable to biological substances.32 The ZP of the ACNPs in Figure 2F was around

33, 50, 51

15


However, these ACNPs


294

−28.8 mV, indicating that a low negative surface charge density, favorable for

295

conjugating to antibody. The polymer dispersity index (PDI) of the naked ACNPs was

296

around 0.1, indicating excellent dispersity of the ACNPs.

297

298

Preparation and characterization of labeled mAbs

299

The stability and reliability of LFAs are mostly based on the quality of the

300

labeled mAbs. The procedure of conjugating ACNPs to mAbs is reported to result in

301

covalent cross-linking by introduction of a bifunctional reagent such as

302

glutaraldehyde.32 After glutaraldehyde was added to a mixture of ACNPs and mAbs, a

303

portion of glutaraldehyde was absorbed the surface of the ACNPs and another part

304

bound covalently to the immobilized mAbs. Since the density of the deposited mAbs

305

was sufficient after 24 h of solubilization, glutaraldehyde covalently conjugated them

306

to each other and formed a rigid shell around an ACNP. The unaltered sites were

307

blocked by added BSA. ACNPs-mAbs conjugates were characterized by TEM

308

(Figure 2B, 2C and 2D), DLS (Figure 2E and Table S3) and ZP (Figure 2F and Table

309

S3). The size of resulting conjugates had increased to slightly larger than the naked

310

ACNPs (Figure 2A), at 172.8 nm for ACNPs-mAb 12E8 (DON), 174.5 nm for

311

ACNPs-mAb 9C7 (T-2) and 163.7 nm for ACNPs-mAb 3D4 (ZEN). Parameters of

312

DLS and ZP values for ACNPs conjugates (Figure 2E, 2F and Table S3) changed

313

after adsorption of mAbs to the ACNPs surface, showing that the mAbs were

314

successfully labeled with naked ACNPs. All PDI values for ACNPs-mAbs were about

16


Page 16 of 41

Page 17 of 41


315

0.1, indicating excellent dispersion of ACNPs-mAbs, and in accordance with naked

316

ACNPs. Thus, ACNPs-mAbs could be used in subsequent studies.

317

Two other labels-mAbs, GNPs-mAbs and QDs-mAbs, were prepared and

318

characterized by the same methods. GNPs of 42.7 nm were chosen since they are

319

known

320

polymer-modified core/multishell QDs of 21.8 nm were employed as fluorescence

321

labels because of their high fluorescence quality and favorable biocompatibility.42

322

Details about the procedure of preparing GNPs-mAbs and QDs-mAbs and identified

323

parameters are presented in (Figure S3 and S4 and Table S3). The date demonstrated

324

the conjugates were successfully prepared.

to

have

good

performance

in

LFIAs.43

Previously

synthesized

325

326

Development of multi-LFAs for mycotoxins

327

After characterization of the immunogens, multi-LFAs for three mycotoxins in

328

buffer were developed. A simple description of the LFA format is given in Figure 1.

329

For a multi-LFA containing one more specific mAbs, a reduction of color intensity at

330

a given test line is caused by an expected mycotoxin. Cross-talk may occur among

331

several pairs of immunoreagents reacting to different analytes in a single device. Thus,

332

before determining sensitivity and specificity of the multi-LFAs, inhibition tests were

333

carried out on three test lines by contrasting changes in color intensity when target

334

mycotoxins were separately added in single format and a three-mycotoxin cocktail

335

was added in the multiple format. The individual mycotoxin standard at a relatively 17



336

high concentration (30 µg/L DON, 20 µg/L T-2 and 5 µg/L ZEN) reduced the color

337

intensity only of the corresponding test line for all three multi-LFAs (Figure 3). The

338

result demonstrated that cross-reaction between different pairs of mAbs-mycotoxins

339

was negligible. Therefore, the coating antigens and mAbs were simultaneously used

340

for multiplexed screening of DON, T-2 and ZEN in a single test strip.

341

The sensitivity of multi-LFAs in buffer was determined by testing mycotoxins

342

standards in concentrations from 0 to 20 µg/L for DON, 0 to 10 µg/L for T-2 and 0 to

343

1 µg/L for ZEN when ACNPs and QDs were used as labels. Concentrations of

344

mycotoxins were 4-times higher when GNPs were used (Figure 4). The vLOD and

345

qLOD for multi-LFAs for each label are in Figure 4 and Table 1. Obtained vLODs

346

were 10 µg/L using ACNPs, 80 µg/L using GNPs, and 20 µg/L using QDs, for DON;

347

and respectively, 5, 40 and 10 µg/L for T-2; and 0.5, 4, and 1 µg/L for ZEN. The

348

qLOD derived from standard curves was 0.5 µg/L using ACNPs, 2.6 µg/L using

349

GNPs and 0.5 µg/L using QDs for DON; and respectively, 0.4, 1.5 and 0.3 µg/L for

350

T-2; and 0.02, 0.2 and 0.1 µg/L for ZEN. Thus, ACNPs provided the highest

351

sensitivity relative to the other two labels, with 2-times or 8-times lower vLOD than

352

QDs and GNPs. ACNPs used as labels in LFAs were preferential to GNPs and

353

generally comparable to QDs for sensitivity which expressed by qLOD (Table 1).

354

Furthermore, the black color derived from ACNPs was clearer than the pink color

355

from GNPs on a background of the white NC membranes (Figure 4A and 4B).

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Although high sensitivity with multi-LFAs for mycotoxins was also achieved by

357

using QDs, the cost and potential toxicology of semiconductor QDs could limit their 18


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wide application in practice. In addition, visual results could not be obtained by the

359

naked eye at nature light when QDs were used as labels. We therefore concluded that

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the price and easy preparation of ACNPs was advantageous over GNPs and QDs for

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LFAs development to enhance detection limit and reduce costs.

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Investigation of the specificity of immunoassays was crucial for assessing results.

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The aim of the study was to evaluate the feasibility of ACNPs for developing LFAs

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for multiple mycotoxins. The specificity of the developed ACNP-LFAs was

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determined. Our examination of CR% was conducted under optimized conditions and

366

results are presented in Table S2. The specificity of the ACNP-LFAs was similar to

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icELISA when using the same pair of antibody and coating antigen: 1600% for

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3-Ac-DON, 16.5% for 15-Ac-DON, and almost 100% for all six ZEN analogs (93.8–

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113.2%) with negligible CR% for HT-2, NIV, FB1, OTA and AFB1 (

Multiplex Lateral Flow Immunoassays Based on Amorphous Carbon

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