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An aptamer-based lateral flow test strip for rapid detection of zearalenone in corn samples. Shijia Wu, Lihong Liu, Nuo Duan, Qian Li, You Zhou, and Zhouping Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05326 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 10, 2018

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Journal of Agricultural and Food Chemistry

An aptamer-based lateral flow test strip for rapid detection of zearalenone in corn samples

Shijia Wu, abc Lihong Liu, b Nuo Duan, ab Qian Li, b You Zhou, b Zhouping Wang abde*

a

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

b

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China c

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

d

International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, China

e

Collaborative innovation center of food safety and quality control in Jiangsu Province, Jiangnan University, Wuxi 214122, China

1

ACS Paragon Plus Environment


1

ABSTRACT

2

An aptamer-based lateral flow test strip was developed for the detection of

3

zearalenone (ZEN). This assay was based on the competition for the aptamer between

4

ZEN and its complementary sequence. Several experimental conditions that could

5

influence sensitivity have been investigated, including the concentration of aptamer

6

and NaCl used in the probe preparation, the mole ratio of streptavidin and biotinylated

7

DNA used in the preparation of test line and control line and the loading quantity of

8

gold nanoparticles-aptamer conjugates (AuNPs-Apt). Under the optimal experimental

9

conditions, we successfully detected ZEN within a detection range of 5 to 200 ng/mL

10

and the visual limit of detection of 20 ng/mL. This aptamer-based strip was

11

successfully applied to the determination of ZEN in spiked corn samples, and the

12

recoveries were from 93.4% to 114.2%. All detections can be achieved within 5 min.

13

The results demonstrated that the developed aptamer-based lateral flow test strip is a

14

potential alternative tool for the rapid and sensitive detection of ZEN.

15

KEYWORDS: lateral flow test strip, aptamer, zearalenone, gold nanoparticles

16

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INTRODUCTION

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Zearalenone (ZEN), a secondary metabolite of Fusarium (Fusarium roseurn), has

19

been regarded as one of the most widely distributed Fusarium toxins in the world.

20

ZEN and its derivatives are mainly found in moldy corn, wheat, barley, oats, sorghum

21

and other cereals and dairy products.1, 2 ZEN has been confirmed as an endocrine

22

disruptor, which can affect the development of internal organs and lead to animal

23

reproductive disorders and digestive system dysfunction,3 and it has the potential to

24

cause substantial economic impacts. Moreover, humans are at risk for ingesting ZEN

25

through the consumption of contaminated food products. Considering the

26

consequences of ZEN on human health, 60 µg/kg of ZEN in wheat and corn has been

27

set as the maximum residue level (MRL) in China. Thus, it is essential and necessary

28

to develop rapid, sensitive and reliable analytical methods for ZEN detection in food

29

products.

30

Currently, instrument methods are often used for the accurate detection of ZEN

31

concentration in samples, such as gas chromatography-mass spectrometry (GC-MS),

32

high-performance

33

chromatography-mass spectrometry (HPLC-MS).4-7 Methods using instruments have

34

the advantages of high sensitivity and specificity; however, they are time-consuming

35

and expensive and they require highly skilled personnel and tedious sample

36

pretreatment. In addition to instrumental methods, immunoassays are powerful

37

bioanalytical techniques for the determination of ZEN, such as chemiluminescence

38

immunoassays,8 fluorescence immunoassays,9,

39

assays (ELISA)11 and lateral flow immunoassays12. Lateral flow immunoassays have

40

attracted much attention due to their advantages of visual observation, simple

41

operations, and cost-effectiveness. They allow direct and rapid analysis of the samples

liquid

chromatography

(HPLC),

10

high-performance

liquid

enzyme-linked immunosorbent

3



42

by the naked eyes without any special instruments. Ji et al developed a ZEN

43

immunoassay strip that can obtain the results in 5 min. The strip was successfully

44

applied to the detection of ZEN in wheat samples with a limit of detection of 15

45

ng/mL.13 Nevertheless, the immunoassays are susceptible to physical and chemical

46

conditions resulting from the unstable antibodies.

47

Aptamers are single-stranded DNA or RNA ligands generated by exponential

48

enrichment (SELEX), which can target a variety of molecules such as cells, metal

49

ions, proteins, bacteria, and viruses.14-16 As a good candidate to antibodies, aptamers

50

have the advantages of a short screening cycle, low cost, easy synthesis, stability and

51

long-term preservation in addition to the high affinity and specificity.17 Recently,

52

many achievements have been made in development and application of aptamers to

53

identify target mycotoxins.

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aptamer-based assays.21-24 However; many aptamer-based assays mainly require a

55

laboratory-based infrastructure, which limits their utility and flexibility. Thus,

56

integrating the advantages of aptamers and lateral-flow technology is a positive step

57

to improve the user-friendliness of aptamers.

18-20

Our group has also reported lots of relative

58

Some strip biosensors based on aptamer-functionalized gold nanoparticles have

59

been successfully developed for thrombin25, adenosine26, and ochratoxin A27 analysis.

60

For the extended application of ZEN aptamer selected by our group, herein, we

61

combined the simplicity, convenience and portability of lateral flow strip and the high

62

affinity, specificity, and stability of aptamers to develop an aptamer-based lateral flow

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test strip for rapid and sensitive detection of ZEN. The developed aptasensor

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exhibited high analytical performance in terms of sensitivity, selectivity, and

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practicability. To the best of our knowledge, this was the first time that an aptamer

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has been applied instead of antibodies to the lateral flow test strip for the detection of

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ZEN.

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MATERIALS AND METHODS

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Chemicals and Materials

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Streptavidin, Emetic toxin, Aflatoxin B1, Ochratoxin A and Zearalenone were

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purchased from Sigma-Aldrich (Saint Louis, MO). All elements of the lateral flow

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test strip (plastic adhesive backing, sample pad and nitrocellulose membrane CN 140,

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and an absorbent pad) were obtained from the Shanghai You Long Biotechnology

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Co., Ltd. (Shanghai, China). The sequences of the ZEN aptamer Apt 1

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(5’-SH-TCATCTATCTATGGTACATTACTATCTGTAATGTGATATGTTTTTTT

76

TTTTTTTTTTTTTTTTTTTTTTT-3’)21 and its complementary sequence DNA 1

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(5’-biotin-CATATCACATTACAGATAGTAATGTACCATAGATAGATGA-3’);

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DNA

79

synthesized by the Sangon Biotechnology Co., Ltd. (Shanghai, China). Tween-20,

80

Ovalbumin (OVA), Tris (2-carboxyethyl) phosphine (TCEP) and other metal salts

81

were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

82

All aqueous solutions were prepared with ultrapure water (≥18 MΩ, Milli-Q,

83

Millipore).

84

Apparatus

2

(5’-bio-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’)

were

85

The size and morphology of the gold nanoparticles (AuNPs) were determined by

86

a JEM-2100 HR transmission electron microscope (TEM, JEOL Ltd., Japan) at 200

87

kV. Ultraviolet-visible (UV–vis) absorption spectra were recorded using a Shimadzu

88

UV-2300 UV–vis spectrophotometer (Shimadzu, Japan). An XYZ 3050 dispensing

89

platform (BioDot, U.S.A.) was used to prepare lateral flow test strip. An MZ-6000

5



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strip reader (Meizheng Biotechnology Co., Ltd., China) was used to analyze the

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aptamer-based lateral flow test strip.

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Preparation of AuNPs

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The synthesis of AuNPs was performed according to the reduction of HAuCl4 by

94

sodium citrate.28 First, 100 mL of 0.01% HAuCl4 solution was added to the bottle

95

with constant stirring and heating. After boiling, 1 mL sodium citrate was added to the

96

bottle immediately. When the color of the liquid changed from gray to wine-red, the

97

solution was heated for another ten minutes and then cooled to room temperature. The

98

prepared AuNPs were stored away from light and stored at 4 °C in the refrigerator.

99

Preparation of Gold Nanoparticles-Aptamer Conjugates (AuNPs-Apt)

100

Briefly, 10 mL of AuNPs solution was concentrated at 10,000 rpm for 10 min to

101

1 mL. 25 µM of 5’-thiolated aptamer was activated by 1 mM TCEP at room

102

temperature for 1 h, and then the aptamer was transferred to the 1 mL prepared

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AuNPs solution to incubate at 4 °C for 24 h. NaCl (1 M) solution was added to the

104

mixture untill the final concentration of NaCl reached 80 mM. Then, the solution was

105

incubated at 4 °C for another 24 h. Excess thiolated aptamer was removed by

106

centrifugation at 10,000 rpm for 20 min, and the conjugates were re-suspended in 0.01

107

M PBS (phosphate buffer saline) (pH 7.4) containing 0.5% PEG, 5% sucrose, 0.25%

108

Tween 20, 0.02% MgSO4, 0.05% (NH4)2SO4, and 1% OVA. Finally, the new

109

AuNPs-Apt was stored in brown bottle at 4 °C for future use.

110

Preparation of Nitrocellulose Membrane

111

In brief, 100 µM of DNA 1 and DNA 2 was incubated separately with 0.125

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mg/mL streptavidin at room temperature for 30 min. Next, the prepared DNA

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1-streptavidin complexes and DNA 2-streptavidin complexes were sprayed on

114

nitrocellulose membrane to form the test line and control line, respectively. The 6


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distance between the two lines was approximately 4 mm. Then, the nitrocellulose

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membrane was dried at 37 °C for 1 h for immobilization.

117

Assembly of the Aptamer-based Lateral Flow Test Strip and Detection

118

Procedures

119

The aptamer-based test strip comprised a plastic adhesive backing, sample pad,

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nitrocellulose membrane, and absorbent pad. The three elements were pasted on the

121

plastic adhesive backing in sequence, overlapping each other approximately 2 mm

122

from each other. Then, the strip was cut to 4 mm and stored out of light for future use.

123

The AuNPs-Apt conjugates and ZEN toxins of different concentrations were mixed

124

and reacted at 30 °C for 10 min. Then the mixture was added to the sample pad. One

125

minute later, 100 µL of PBS (pH 7.4) was added to the sample pad to wash away the

126

excess AuNPs-Apt, and the results were observed three minutes later.

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Sample Assay Procedures

128

We applied the test strip to analyze the ZEN contamination in corn samples. The

129

corn samples were smashed and dissolved in a 70% ethanol-water solution. The liquid

130

was centrifuged at 4,000 rpm for 1 min, and then the supernatant was diluted with

131

PBS (pH 7.4). Different amounts of ZEN were added into the solution, and the final

132

concentrations of ZEN were 0, 5, 50, 100, 150, 200, 300 and 400 ng/mL. The solution

133

was added to the prepared test strips, and one minute later, 100 µL of PBS (pH 7.4)

134

was added to the sample pad to wash away the excess AuNPs-Apt. Three minutes

135

later, the results were recorded.

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RESULTS AND DISCUSSION

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Principles for the Aptamer-Based Lateral Flow Test Strip

138

The principle for the aptamer-based lateral flow test strip was based on the

139

competition for AuNPs-Apt between the DNA 1 on the test line and ZEN in the 7



140

samples (Figure 1). In the absence of ZEN, AuNPs-Apt combined with DNA 1 on the

141

test line and DNA 2 on the control line by complementary base pairing, resulting in

142

two lines on the strip. In the presence of ZEN, AuNPs-Apt preferred to bind with

143

ZEN, weakening the combination of AuNPs-Apt and DNA 1. As a result, the color

144

intensity of test line was decreased or diminished. The color intensity of the test line

145

depended on the concentration of ZEN in the solution. Moreover, the capture of

146

AuNPs-Apt and DNA 2 on the control line occurred regardless of whether there was

147

ZEN in the solution, leading to constant color intensity in the control line. In this

148

study, the minimum concentration of ZEN showing a distinguishable color difference

149

from the negative control was defined as the detection limit.

150

Optimization of the Binding Conditions of Aptamer and AuNPs

151

When AuNPs and aptamer exist simultaneously in a solution, NaCl can help to

152

form a stable Au-S bond between the thiol group tagged to the aptamer and the

153

AuNPs to increase the load capacity. To investigate the influence of NaCl, different

154

concentrations of NaCl were added to the solution of AuNPs and aptamers. After

155

aging and incubation, the mixture was centrifuged and the supernatant was measured

156

by ultraviolet spectrophotometer (Figure S1). With the increase of NaCl concentration

157

from 0 to 100 mM, the absorbance intensity of the supernatant decreased, signifying

158

that more aptamers had attached to the surface of the AuNPs. When the concentration

159

of NaCl reached 80 mM, the load capacity of aptamer was almost saturated; thus, 80

160

mM of NaCl was chosen to be used in the subsequent study.

161

In the process of preparing AuNPs-Apt, the addition of NaCl can increase the

162

load capacity of the aptamer and help to form a more stable connection. Meanwhile,

163

the Na+ and Cl- will destroy the ionic environment and lead to the aggregation of

164

AuNPs. In addition, the aptamer on the surface of AuNPs can prevent them from 8


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aggregation. Sufficient aptamer can make the AuNPs-Apt solution remain a bright red

166

color; otherwise, it will aggregate into purple.29,30 Therefore, under the NaCl

167

concentration of 80 mM, different final concentrations of aptamer (0~1000 nM) were

168

used to study the binding condition between aptamer and AuNPs, respectively. TEM

169

images of the AuNPs suspension were recorded (Figure 2). The AuNPs aggregated to

170

various degrees under the low concentration of aptamer. When the concentration of

171

aptamer was up to 600 nM, the particles demonstrated a good dispersion, and the

172

solution was a bright red color. Thus, the final concentration of aptamer was set as

173

600 nM.

174

Optimization of the Performance of Control Line and Test Line

175

Nucleic acid can be washed away easily by the sample flow if sprayed onto the

176

nitrocellulose membrane directly because it has no specific binding force to

177

nitrocellulose membrane. Therefore, streptavidin was set as a bridge, which can be

178

immobilized on the nitrocellulose membrane by electrostatic adsorption and linked

179

biotin-modified DNA through the specific reaction of streptavidin and biotin. The

180

conjugation of streptavidin and biotin-modified DNA 1 and DNA 2 impacted not only

181

the immobilization efficiency of DNA 1, DNA 2 onto the nitrocellulose membrane

182

but also the hybridization efficiency between DNA 1 and DNA 2 and AuNPs-Apt. To

183

optimize the performance of the test line constructed by streptavidin and DNA 1 and

184

control line constructed by streptavidin and DNA 2, the molar ratios of streptavidin

185

and DNA 1 and DNA 2 were studied. Streptavidin (0.125 mg/mL) was mixed with

186

DNA 2 (100 µM) in molar ratios from 2:1, 1:1, 1:3, 1:6, 1:9, 1:15 to 1:20. After being

187

sprayed onto the nitrocellulose membrane, 30 µL of AuNPs-Apt was added to the

188

sample pad. As shown in Figure 3, a good performance of color intensity of the

9



189

control line was shown at the ratio of 1:6. The same results were shown in the test line.

190

Thus, the molar ratio between streptavidin and DNA 1 and DNA 2 was set at 1:6.

191

Optimization of the AuNPs-Apt Loading Quantity

192

To improve the sensitivity of ZEN detection, the concentration of AuNPs-Apt

193

was optimized systematically. Different concentrations of AuNPs-Apt (60, 30, and 10

194

µL) were used to conjugate with ZEN (final concentration of 50 ng/mL); the results

195

are recorded in Figure 4. Under the high concentration of AuNPs-Apt, it preferred to

196

bind with ZEN and the remaining AuNPs-Apt hybridized with the test line, causing

197

false negative results. Under the low concentration of AuNPs-Apt, the color intensity

198

of both the test line and the control line decreased or even disappeared. The most

199

obvious and appropriate color intensity of the lines was obtained in the second group.

200

Therefore, 30 µL of AuNPs-Apt was selected in the subsequent experiments.

201

Sensitivity and Specificity of the Aptamer-based Lateral Flow Test Strips

202

Under the optimal conditions, the developed aptamer-based lateral flow test strip

203

was used to detect ZEN in the sample solution. Various concentrations of ZEN were

204

mixed with AuNPs-Apt to final concentrations of 0, 5, 20, 50, 100, 200, 300, and 500

205

ng/mL. The samples were introduced to the prepared test strip, and the results were

206

recorded. As shown in Figure 5a~h, with the increase in ZEN concentration in the

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detection solution, the color intensity on the test line decreased gradually. The

208

intensity of the color on the test line was obviously weaker than the negative at the

209

concentration of 20 ng/mL. Therefore, 20 ng/mL can be treated as the visual limit of

210

detection (LOD) of the aptamer-based test strip for ZEN detection. The samples were

211

further analyzed on an MZ-6000 strip reader. As shown in Figure 5i, the test line was

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gradually decreased with the increase of ZEN concentration. A calibration plot 10


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between the relative intensity (test line value/control line value) and the logarithm of

214

ZEN concentration displayed a good linear relationship from 5 to 200 ng/mL and fit

215

the linear regression equation y=-0.169x+0.516, R2=0.9976 with the detection limit

216

of 5 ng/mL (Figure 5j).

217

All detections can be achieved within 5 min. Compared with other detection

218

methods for ZEN (Table S1), although our method was not the best in detection

219

sensitivity, it reached the expected effect required from maximum residue levels of 60

220

µg/kg in China. Moreover, the aptamer-based lateral flow strip is very simple and

221

convenient to use, and it is especially suitable for on-site inspections.

222

The specificity of the aptamer-based lateral flow strip was evaluated using four

223

different toxins, DON, OTA, fumonisin B1 (FB1) and aflatoxin B1 (AFB1). The

224

concentration of ZEN was chosen to be 200 ng/mL, and the other toxins were set at

225

500 ng/mL. As shown in Figure S2, there was only one positive result with ZEN. The

226

results demonstrate that the high specificity of the aptamer-based lateral flow strip

227

towards ZEN.

228

Analysis of ZEN in Corn Samples with the Aptamer-based Test Strips

229

The treated sample solution was added to the prepared test strips, and the results

230

are recorded in Table 1. The sample with no ZEN showed a negative result on the

231

strip membrane. All of the samples containing ZEN above 5 ng/mL demonstrated

232

positive results, and the color intensity of the test line became weaker with increasing

233

concentrations of ZEN. When the concentration was greater than 200 ng/mL, the test

234

line disappeared. The detection results of ZEN were further calculated by the reader.

235

The obtained results showed the successful application of the developed

236

aptamer-based test strip to test corn samples.

237 11



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Stability of the Aptamer-based Test Strips

239

We also evaluated the stability of the aptamer-based test strips for ZEN. All

240

strips were stored for two months at room temperature under dark and dry conditions,

241

and then used to detect samples containing 50 ng/mL of ZEN. Neither the color

242

intensity nor the sensitivity showed any difference from the results obtained with

243

newly prepared strips. This means that the aptamer-based test strip remains stable

244

within two months.

245

In summary, a simple and sensitive aptamer-based lateral flow test strip for ZEN

246

was successfully developed using the competitive format. Unlike a previously

247

reported lateral flow test strip which used antibodies, we utilized aptamers as the

248

recognition elements. The features of aptamers, such as high affinity and specificity,

249

inexpensive and simple synthesis, easy modification and stability during storage,

250

enabled the lateral flow test strip to be more cost-effective and stable. Moreover, the

251

aptamer-based lateral flow test strip also maintained the rapid and simple advantages

252

of test strips. Under the optimized conditions, the visual limit of detection of the strip

253

was as low as 20 ng/mL, and the linear range was from 5 to 200 ng/mL in buffer

254

condition. The detection can be performed within 5 min. The developed strip was

255

successfully applied to the detection of ZEN in corn samples. The aptamer-based

256

lateral flow test strip has overcome the limitation of aptasensors in laboratory-based

257

infrastructure. It is useful as a point-of-use product so long as visual observation is

258

available by the naked eyes without the need for a laboratory. Our novel

259

aptamer-based lateral flow test strip can provide rapid, simple, and sensitive results

260

for numerous food contaminants and multitudinous sample inspection.

261 262

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ASSOCIATED CONTENT

264

Supporting information

265

This material is available free of charge via the Internet at http://pubs.acs.org.

266

Optimization of the final concentration of NaCl to stabilize the AuNPs-Apt solution

267

determined by UV/vis spectra (Figure S1). Specificity of the aptamer-based lateral

268

flow test strip for ZEN (Figure S2). A summary of different detection methods for

269

ZEN (Table S1)

270

AUTHOR INFORMATION

271

Corresponding author

272

* E-mail: [email protected], [email protected]

273

Funding

274

This work was partially supported by China Postdoctoral Science Foundation

275

(2016T90430, 2015M580402), Key Research and Development Program of Jiangsu

276

Province BE2016306, Natural Science Foundation of Jiangsu Province BK20140155.

277

Notes

278

The authors declare no competing financial interest.

279

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Figure caption Figure 1. Aptamer-based lateral flow test strip. Structure of test strip (a). Negative test: in the absence of ZEN (b). Positive test: in the presence of ZEN (c). Figure 2. TEM images of the AuNPs-Apt supernatant. The concentrations of the aptamer were 0 (a), 200 (b), 400 (c), 600 (d), 800 (e), and 1000 nM (f). Figure 3. Optimization of the molar ratio of streptavidin and DNA 2. The ratios of streptavidin to DNA 2 were 2:1 (a), 1:1 (b), 1:3 (c), 1:6 (d), 1:9 (e), 1:15 (f) and 1:20 (g). Figure 4. Optimization of the loading quantity of AuNPs-Apt. The loading quantities test were 60 (a), 30 (b), and 10 µL (c). In each group, the left one was the negative control, and the right one was the experimental treatment. Figure 5. The detection results of different concentrations of ZEN: a (0), b (5 ng/mL), c (20 ng/mL), d (50 ng/mL), e (100 ng/mL), f (200 ng/mL), g (300 ng/mL), and h (500 ng/mL). 3D curves of different concentrations of ZEN obtained from the strip reader (i), Calibration curve of the relative intensity (T/C) versus the logarithm of the ZEN concentration (j). Table 1 Detection results in corn samples with spiked ZEN

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Table 1 Detection results in corn samples with spiked ZEN

Sample

Spiked Concentration

Visual results

of ZEN (ng/mL)

Detection results of

Recovery

instrument (ng/mL)

rate

1

0

Negative

0

/

2

5

Positive

5.71

114.2%

3

50

Positive

53.32

106.6%

4

100

Positive

93.37

93.4%

5

150

Positive

140.13

93.4%

6

200

Positive

191.17

95.6%

7

300

Positive

/

/

8

400

Positive

/

/

24


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Graphic for table of contents

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Aptamer-Based Lateral Flow Test Strip for Rapid Detection of

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