Selection of a DNA Aptamer against Zearalenone and Docking

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Selection of a DNA aptamer against zearalenone and docking analysis for highly sensitive rapid visual detection with a label-free aptasensor Yuanyuan Zhang, Taofeng Lu, Yue Wang, Chenxi Diao, Yan Zhou, Lili Zhao, and Hongyan Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03963 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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Journal of Agricultural and Food Chemistry

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Selection of a DNA aptamer against zearalenone and docking analysis for highly sensitive

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rapid visual detection with label-free aptasensor

3 4

Yuanyuan Zhang#1, Taofeng Lu#1, Yue Wang1, Chenxi Diao1, Yan Zhou1, Lili Zhao1 and

5

Hongyan Chen*1

6 7

Yuanyuan Zhang#, [email protected], Heilongjiang Provincial Key Laboratory

8

of Laboratory Animal and Comparative Medicine, Laboratory Animal and Comparative

9

Medicine, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research

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Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

11

Taofeng Lu#, [email protected].

12

Yue Wang, [email protected].

13

Chenxi Diao, [email protected].

14

Yan Zhou, [email protected].

15

Lili Zhao, [email protected].

16

Corresponding Author: Hongyan Chen*, [email protected], Tel./Fax: +86-451-5105-

17

1790.

ACS Paragon Plus Environment


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Selection of a DNA aptamer against zearalenone and docking analysis for highly sensitive

19

rapid visual detection with label-free aptasensor

20 21

Yuanyuan Zhang#1, Taofeng Lu#1, Yue Wang1, Chenxi Diao1, Yan Zhou1, Lili Zhao1 and

22

Hongyan Chen*1

23 24

ABSTRACT: Contamination of feed with zearalenone (ZEN) presents a significant risk to

25

animal health. Here, a visible, rapid, and cost-effective aptamer-based method is described

26

for the detection of ZEN. After 8 rounds of SELEX (systematic evolution of ligands by

27

exponential enrichment) with an affinity-based monitor and counter-screening process, the

28

ssDNA aptamer Z100 was obtained, which had high affinity (dissociation constant = 15.2±

29

3.4 nM) and good specificity. Docking analysis of Z100 indicated that non-covalent bonds

30

(π-π interactions, hydrogen bonds, and hydrophobic interactions) helped ZEN to anchor in

31

the binding sites. Finally, a label-free detection method based on gold nanoparticles and

32

Z100 at 0.25 μM was developed for ZEN determination. Excellent linearity was achieved

33

and the lowest detection limit was 12.5 nM. This rapid and simple method for ZEN analysis

34

has high sensitivity and can be applied for on-site detection of ZEN in animal feeds.

35

KEY WORDS: zearalenone, aptamer, Lable-free aptasensor, docking analysis, feed

36

detection

37 38


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1. Introduction

40

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin, primarily produced by

41

Fusarium graminearum and Fusarium culmorum, which grows on cereal grains and is

42

associated with reproductive disorders of farm animals (swine, cattle, and sheep)1, reduced

43

fetal weight and embryonic survival of rats 2, premature puberty syndrome, endometrial

44

hyperplasia and neoplasia, as well as cervical cancer and hyperoestrogenic syndromes in

45

humans 1. To protect public health, maximal limits of ZEN residues in foodstuffs and feed

46

have been established by the governments of many countries 3-7. Traditional methods for

47

quantification of ZEN, which include liquid chromatography-mass spectrometry,

48

immunochemical

49

chromatography, and high-performance liquid chromatography

50

the rapid, sensitive, and economical analysis of numerous samples. Therefore, it is

51

necessary to develop such methods for the determination of ZEN residues in agricultural

52

commodities.

approaches,

fluorescence

polarization 8-9,

immunoassay,

gas

cannot be adapted for

53

Aptamers are highly stable oligonucleotides or peptides that bind to target molecules

54

with high specificity 9, which were considered as the most potential antibodies alternative.

55

Many techniques for aptamer selection were developed, for instance, magnetic beads,

56

capillary electrophoresis, whole cell- systematic evolution of ligands by exponential

57

enrichment (SELEX), surface plasmon resonance or flow cell SELEX, robotic SELEX,

58

microfluidic-based SELEX next generation sequencing-based methods and so on10. To

59

date, the biosensing platform based on aptamers achieved substantial development 11-14. A



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number of aptasensors have been developed for the food safety-related targets, such as

61

antibiotics, mycotoxins, heavy metals, pesticides, some plastic polymer products used for

62

food packaging and foodborne pathogens10. Aptamer-based detective technology is widely

63

used for the detection of mycotoxins, especially ochratoxin

64

aflatoxin (AFB1) 20-24, deoxynivalenol 18-19 and T-218-19. Enzyme-linked aptamer assays25

65

and enzyme-linked immunosorbent assay25 were instituted on account of the colorimetric

66

method. Wang et al.

67

50.5 ± 5.4 nM] for an enzyme-linked oligonucleotide assay to measure ZEN concentrations

68

in corn. In addition, ZEN aptamers 27 have been developed as highly sensitive fluorescence

69

sensors 9 and uesd in novel aptasensor assays 8 and lateral flow test strips 28. As label-free

70

aptamer-based biosensors, gold nanoparticles (AuNPs) are highly specific and sensitive for

71

the visualization and on-site detection of target molecules 29-30.

26

15-17,

fumonisin B115,

18-19,

used selected aptamer [equilibrium dissociation constant (Kd) of

72

Molecular docking, an in silico strategy for drug discovery, has been used to study the

73

binding mechanism between aptamers and ligands 31. Novel sensitive enzymes have been

74

employed for the detection of organophosphorus pesticides

75

enzyme

76

evaluate the coordination between computational models and experimental analyses

77

Molecular docking provides insights into how receptor interacts with a ligand. AutoDock

78

is an automated molecular docking software package to quantitatively evaluate the binding

79

of substrates or drug candidates 34.

80

32

31.

For example, the gyrase

can be used to determine the binding area within the aptamer structure to 33.

In this study, a ZEN binding aptamer was developed by immobilization-free selection


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81

with affinity-based monitoring. The unmodified AuNP-based label-free aptasensors were

82

developed for the rapid detection of contamination of ZEN residues in feeds.

83 84

2. Materials and Methods

85

2.1 Reagents and chemicals

86

ZEN, α-zearalenol (α-ZON), β-zearalenol (β-ZON), AFB1, and T-2 (purity > 98 %

87

respectively) were purchased from Pribolab Pte. Ltd. (Singapore). Fumonisin B1 (FB1)

88

(purity > 98 %) was purchased from Aladdin Biochemical Technology Co. Ltd. (Shanghai,

89

China). All other chemicals for preparing buffers were obtained from Sigma-Aldrich (St.

90

Louis, MO, USA) if not stated otherwise. The PCR components were purchased from

91

TransGene Biological Science & Technology Company (Beijing, China). Water was

92

deionized and purified using a Millipore system (Millipore, Bedford, MA) for preparing

93

solutions.

94 95

2.2 Capture-SELEX Library, capture oligos (COs) and primers

96

A 98-base random ssDNA oligonucleotide library (OL), as described by Reinemann et al.

97

35

98

purified by high-performance liquid chromatography. The oligonucleotide of library

99

contains sequences of 10 and 40 nucleotides (nt) flanked by defined primer-binding sites

100

(18 nt each) at the 3’ and 5’ ends with a specific 12-nt docking sequences located between

101

two random sequence. The CO

(Table 1), was synthesized by Comate Bioscience Co., Ltd. (Changchun, China) and

35

was developed that consists of four parts (5’ to 3’): a



102

biotin, three additional nucleotides (G, T, and C), a hexaethylene glycol spacer (HEGL),

103

and a docking sequence. The primers 5’-fluorochrome (FAM) modified F (FF) and 5’-

104

poly-dA20-HEGL modified R (FR) were used to separate the double-stranded PCR

105

products (Table 1). PCR amplification from the last selection round was performed using

106

forward (F) and reverse (R) primers for cloning and sequencing. (Table 1). Table 1 Sequences and modifications of the library and primers

107

Library and primers

Sequences 5’–3’ and modifications

OL

ATACCAGCTTATTCAATT-N10-TGAGGCTCGATC-N40ACAATCGTAATCAGTTAG

CO

Bio-GTC-HEGL-GATCGAGCCTCA

FF

FAM- ATACCAGCTTATTCAATT

FR

Poly-dA20-HEGL- CTAACTGATTACGATTGT

F

ATACCAGCTTATTCAATT

R

CTAACTGATTACGATTGT

108

N10 and N40: random regions, FAM: fluorochrome, Poly-dA20: 20 adenine, HEGL:

109

hexaethylene glycol spacer, Bio: Biotin.

110 111

2.3 Monitoring of the Capture-SELEX process

112

A schematic diagram of the SELEX process, based on the descriptions of Stoltenburg et al.

113

35 and Nathalie et al. 36, is shown in Figure 1. Dynabeads (MyOne Streptavidin C1, diameter,

114

1.0 μm) were used to immobilize the CO of biotin modified oligos. The beads were washed


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three times with 2× binding and washing (BW) buffer and then resuspended with an equal

116

volume CO solution. After 1 h of incubation at room temperature (RT) with shaking, the

117

beads were magnetically separated, washed three times with 500 μL of binding and

118

washing buffer, and stored in binding buffer (50 mM Tris, 100 mM NaCl, 5 mM KCl, 2

119

mM MgCl2, 1 mM CaCl2, 0.02 % vol Tween 20, pH 7.4) at 4°C at a final concentration of

120

109 beads/mL.

121

The beads with CO were incubated in Capture-SELEX procedure of binding buffer

122

overnight with mild shaking at room temperature, together with the oligonucleotide library

123

of 2 nM for the first round and the following quantity of the selected oligos from the

124

previous round (folded at 90 °C for 10 min, 4 °C for 10 min, balanced at room temperature

125

for a few minutes). Seven washes with 500 μL of binding buffer were necessary to remove

126

the unbound beads from the ssDNA-bead complexes.

127

Positive and negative selection strategies were chose to improve the efficiency. As the

128

selection target in the first round of Capture-SELEX process, 1mM sterilized ZEN was

129

mixed with ssDNA-bead complexes in 300 μL of binding buffer. After incubating for 1 h

130

at temperature, the beads from the first round were removed and the pool of oligo-riched

131

supernatant was amplified by PCR in 20 parallel reactions. For amplification, 0.5 μL of the

132

pooled sample, as a template, was combined with 0.5 μL of a 10 μM solution of the forward

133

and reverse primers (FF and FR), 10 μL of 2×Taq PCR StarMix with Loading Dye (Genstar,

134

Beijing, China) and 8.5 μL of ddH2O. A double-stranded DNA (dsDNA) pool was obtained

135

by PCR (3 min at 95 °C, then 26 cycles at 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s,



136

and a final extension at 72°C for 5 min). The two strands of the PCR products were

137

denatured by heating and separated by polyacrylamide gel electrophoresis. After

138

visualization, the ssDNA-FAM strand was recovered and purified as the selection pool for

139

the second round.

140

In the following rounds of negative selections, 108 Dynabeads beads were used for

141

capture oligos by incubating with the selection pool form previous round. Five structural

142

analogue mycotoxins (α-ZON, β-ZON, AFB1, T-2 and FB1) were used as a negative

143

selection mixture for aptamer selection to increase the specificity and mixed to a final

144

concentration of 1 mM. Instead of ZEN, negative selection mixture was incubated with the

145

DNA-bead-complexes. The supernatant containing oligos binding to negative selection

146

mixture was discarded and the DNA-bead-complexes were washed six times with binding

147

buffer. The remaining oligos bound to the beads were heated and eluted as the pool for

148

PCR and later steps, as round 1.

149

The ZEN-specific aptamer pool was obtained after eight rounds of the Capture-

150

SELEX procedure. The methods adopted in this experiment for identification of the

151

aptamer sequences were based on the descriptions by Lu et al 29. The purified dsDNA of

152

PCR product was subcloned into the plasmid pMD-18T, which was transformed into

153

competent Escherichia coli DH5α cells. In total, 105 clones were randomly selected,

154

purified, and sequenced by Sangon Biotech Co. Ltd. (Shanghai, China). The identified

155

candidate aptamers were named as Z1 to Z105.

156

2.4 Binding assays, specificity and SELEX process monitoring by fluorescence


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26

157

With modifications, fluorescence

was used to detect the binding affinity of objective

158

aptamers. A microtiter plate was coated with 50 μL (5 μg/mL) of bovine serum albumin-

159

conjugated ZEN (ZEN-BSA) (Pribolab Pte. Ltd.) and incubated at 4 °C overnight. After

160

washing three times with phosphate-buffered saline, the coated wells were blocked by

161

incubating with 5% BSA for 1 h at 37°C. After washing, 90 μL (0- 2 μM) of FAM-ssDNA

162

aptamers were added to the coated wells and the plate was incubated for 1 h at 37°C. After

163

washing three times, fluorescence was measured with a microplate reader (Bio-Tek) at a

164

Fluorescence Intensity (a.u.). Alpha-ZON-BSA, β-ZON-BSA, AFB1-BSA, T-2-BSA, and

165

FB1-BSA were used to determine the specificity of the selective candidate aptamers. The

166

obtained ssDNA selection pools (- 200 nM) were monitored in round 8. SigmaPlot software

167

(version 12.0; Systat Software Inc., Chicago, IL, USA) was used to analyze the obtained

168

data and estimate the Kd values of the aptamers.

169 170

2.5 Molecular docking

171

A molecular docking study was performed to investigate the binding mode between the

172

ZEN and aptamers using the AutoDock Vina open-source program (ver. 1.1.2)

173

Mfold web server (http://www.bioinfo.rpi.edu/applications/mfold/) was used for prediction

174

of secondary structure of the linear ssDNA 33. The file of Vienna output format (dot-bracket

175

notation)

176

(http://biophy.hust.edu.cn/3dRNA) of the aptamer 33. The structure of the ZEN molecule

177

with the proper two-dimensional (2-D) orientation was drawn using ChemBioDraw Ultra

was

used

to

construct

the

three-dimensional


(3-D)

37.

The

structure


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14.0 software (Adept Scientific PLC, Luton, UK) and the structure was checked for

179

drawing errors. The energy of ZEN was minimized using ChemBio3D Ultra 14.0 software

180

(PerkinElmer, Inc., Waltham, MA, USA). The AutoDockTools 1.5.6 package

181

employed to generate the docking input files. For Vina docking, the default parameters

182

were used, unless otherwise indicated. The best-scoring pose, as judged by the Vina

183

docking score, was chosen and visually analyzed using PyMoL 1.7.6 software

184

(http://www.pymol.org/ ).

38

was

185 186

2.6 ZEN detection and synthesis of citrate-protected AuNPs

187

AuNPs were synthesized using the classical citrate reduction method29. Briefly, sodium

188

citrate solution (10 mL, 38.8 mM) was rapidly injected to boiling aqueous solution of

189

HAuCl4 (100 mL, 1 mM). After boiling for 20 min, the reaction solution was cooled to RT

190

and stored at 4 °C for further use. The final AuNPs were characterized using a transmission

191

electron microscope29 (H-7650, Hitachi Ltd., Tokyo, Japan).

192

A 25 μL aliquot of the aptamer solution (0.125, 0.25, 0.5 and 1.0 μM) was mixed with 50

193

μL of an aqueous solution of ZEN (4, 8, 16, 32, 64 and 128 ng/mL) and added to the wells

194

of a microplate, which was incubated at room temperature for 2 min. Afterward, 50 μL of

195

AuNPs was added, homogenized, and left standing for 2 min. Then, 5 μL of 2 M NaCl

196

were added to terminate the reaction. After the mixed solution was equilibrated for 2 min,

197

the absorption spectrum at wavelengths of 450- 750 nm within 30 min was measured with

198

an EnSpire automatic microplate reader (PerkinElmer, Inc.). All assays were performed at


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199

RT.

200

2.7 Samples analysis

201

A spiking study, conducted with corn powder purchased from a local supermarket and

202

commercial ZEN-free mouse feed, was used to demonstrate the practicability of the visual

203

label-free aptasensor bioassay for the detection of ZEN. The samples were dried overnight

204

in an incubator at 60°C. The extraction process was performed as described by Wang et al.

205

26.

206

μg/kg) and the mixture was incubated overnight at RT. The next day, the mixture was

207

assayed using an ssDNA-based fluorescence. The samples were extracted in 40 mL of

208

methanol/water (70:30, v/v) and diluted with sterilized water. ZEN recovery from the

209

supernatants was determined.

Then, 10 g of the sample were added to standard concentrations of ZEN (16, 32 and 128

210

Three kinds of on-site contaminated feeds from nine farms located in Heilongjiang

211

province were collected, including condensed feeds of lactation cow (represented A, B,

212

and C samples), complete feeds for pregnant sows (samples D, E, and F), and complete

213

feeds for layer chickens (samples G, H, and I). The extraction and detection processes are

214

described above. Meanwhile, on-site contaminated feeds were determined using two

215

commercially

216

(RIDASCREEN Zearalenone, R-Biopharm AG, Darmstadt, Germany; Huaan Magnech

217

Bio-Tech Co., Ltd., Beijing, China). All samples were analyzed in triplicate.

218

3. Results and Discussion

219

3.1 Aptamer selection procedure

available

enzyme-linked

immunosorbent


assay

(ELISA)

kits


220

The Capture-SELEX process (Figure 1) is suitable and efficient method for the selection

221

of small molecules that are difficult to immobilize 35.

222 223

Figure 1 Capture-SELEX strategy

224

3.1.1 Optimization of PCR annealing temperature

225

The annealing temperature used for the PCR has a substantial effect on amplification. Thus,

226

annealing temperatures from 55°C to 64°C were tested to optimize the system. Finally,

227

61°C was chosen as the optimal annealing temperature (Figure S1). A maximum yield of

228

dsDNA was obtained after 30 cycles of amplification.

229

3.1.2 Selection of aptamers against ZEN


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The binding affinity of ZEN-BSA after 1-8 rounds was evaluated using the fluorescence

231

method. As shown in Figure 2, the Fluorescence Intensity values indicate that affinity

232

increased more than 10-fold after eight rounds of screening, with maximum values reached

233

after the seventh round. From round three, the addition of the negative selection mixture

234

sharply increased the selective pressure and improved the efficiency and accuracy of

235

screening. Fluorescence Intensity （a.u.）

230

800 600 400 200 0 1

2

3

4 5 Selection round

6

7

8

236 237

Figure 2 The binding affinity of 1-8 selected rounds by the fluorescence method.

238

In total, 105 clones were selected and sequenced after obtaining the enriched dsDNA

239

pool. FAM-labeled ssDNA aptamers, 98 nt in length, were generated by PCR, separated

240

by polyacrylamide gel electrophoresis, recovered, and purified using the 105 clones as

241

templates. The affinities of the 13 sequences (sequences in Supplementary Table 1), the R2

242

values ranged from 0.92 to 0.98, were considered reliable (Table 2). The lowest Kd value

243

compared

244

(ATACCAGCTTATTCAATTCTACCAGCTTTGAGGCTCGATCCAGCTTATTCAAT

245

TATACCAGCTTATTCAATTATACCAGCACAATCGTAATCAGTTAG) might have

to

other

aptamers


of

Z100


higher

affinities

for

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246

significantly

ZEN.

Z93

247

(ATACCAGCTTATTCAATTCAGAAAAGAGTGAGGCTCGATCTGTAGGATCGAT

248

TAAAATTAACAAATAATGTGTACCATGGACAATCGTAATCAGTTAG) with low

249

binding affinity was as negative control for further analysis. Table 2 The affinities of 13 sequences

250

Number

Aptamer

Kd (nM)

R2

1 2 3 4 5 6 7 8 9 10 11 12 13

Z5 Z12 Z18 Z21 Z23 Z29 Z55 Z61 Z82 Z93 Z100 Z101 Z105

234.8±51.9 92.9±46.8 224.5±48.7 220.3±65.1 49.0±14.9 334.5±105.1 246.7±70.6 94.7±29.9 148.2±79.3 440.0±129.5 15.2±3.4 37.9±7.0 174.6±46.7

0.9876 0.9282 0.9878 0.9791 0.9709 0.9758 0.9783 0.9658 0.9307 0.9842 0.9839 0.9876 0.9799

251

Specificities of 13 ssDNA aptamers (Figure S2) for ZEN-BSA as well as α-ZON-BSA,

252

β-ZON-BSA, AFB1-BSA, T-2-BSA, and FB1-BSA were determined using fluorescence.

253

All of the 13 aptamers showed binding capacity to ZEN, with aptamer Z100 appearing to

254

possess optimal specificity against ZEN (Figure 3). Z93 with low binding affinity and

255

specificity was as negative control for further binding mode analysis.



Relative fluorescence intensity ration (%)

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256

34 32 ZEN

30

FB1

28

AFB1

26

T-2

24

α-ZON β-ZON

22 20 Z100

Z93

257

Figure 3 Determination of the specificity of aptamers by fluorescence. Each data point

258

represents the average ± the standard deviation of three replicates

259

3.2 Aptamer characterization and sequence Analysis

260

The binding affinities of two aptamers (Z100 and Z93) were assessed by measuring the Kd

261

values. The aptamer Z100 had a highest binding affinity with a lowest Kd value (15.2± 3.4

262

nM) as compared to Z93 (440.0± 129.5 nM) (Figure 4a).

263

A three-step procedure [acquisition of structural information for aptamer RNA 39, docking

264

of the molecule into the pocket of the target, and scoring of the molecule docking 40] is

265

generally used to evaluate the affinity of a ligand to the target receptor. The 2-D structures

266

of the candidate aptamers were predicted (Figure 4b). A stem-loop was the most typical

267

structure. As already reported, stem-loop structures of aptamers play a pivotal role in the

268

binding of ligands and receptors 26-27, 29. The binding sites of ZEN targeted both stem and

269

loop in the 2-D structure of Z100 and mainly the loop structure of Z93, which had a more

270

compact and simple stem-loop motif and may be favorable to affinity (Figure 4b).



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271

To investigate the binding mode of ZEN in the binding pocket of the aptamers, ZEN

272

was docked in the binding sites of aptamers Z100 and Z93. As shown by the results in

273

Figure 4c, ZEN adopted a compact conformation to bind inside of the aptamer pocket. The

274

value of exhaustiveness was set to 20. The search grid of Z100 and Z93 were identified as

275

center_x: 56.905, center_y: -102.612 and center_z: -2.803, center_x: 66.946, center_y: -

276

49.789 and center_z: -36.581, which were indicated different docking sites.

277

The designs of the binding domains of the aptamers were based on structural

278

compatibility, aromatic ring stacking, electrostatic and Vander Waals interactions,

279

hydrogen bonding, or a combination of these requirements

280

hydrophobic active pocket of Z100, which was surrounded by the side chains of the

281

nucleotides U-71, U-72, A-38, U-39, A-69, C-68 and U-35, and formed stable hydrophobic

282

bonds. Detailed analysis showed that the phenyl group of ZEN formed π-π interactions

283

with the U-72 nucleotides. Between ZEN and Z100, one of the ester groups of the ZEN

284

formed hydrogen bonds with the polar hydrogen atoms of the nucleotide G-37, with a bond

285

length of 3.3 Å. Two of the hydroxyl group of ZEN formed hydrogen bonds with the

286

carbonyl “O” of nucleotide G-37, with bond lengths of 2.1 Å (Figure 4c). ZEN was located

287

at the hydrophobic pocket in Z93, which was surrounded by the side chain of the

288

nucleotides A-60, A-25, G-28 and U -13 forming a stable hydrophobic binding. Between

289

ZEN and Z93, one of the hydroxyl group of the ZEN formed hydrogen bonds with the

290

carbonyl “O” of the nucleotide U-13 with a bond lengths of 3.7 Å. One of the carbonyl “O”

291

of the ZEN formed hydrogen bond with the hydroxyl “H” of the nucleotides A-15 with the


41.

ZEN was located in the

Page 17 of 33


292

bond lengths of 3.5 Å. All of these interactions helped ZEN to anchor in the binding sites

293

of the aptamers.

294

Molecular docking methods have been used to identify ligands, predict the bond

295

conformation, and specify the bonding mechanism between small molecular compounds

296

and aptamers. After molecular docking of aptamers with the ligands ZEN, the binding free

297

energies of Z100 and Z93 with ZEN, as evaluated with AutoDockTools 1.5.6, were -9.6

298

and -5.8 kcal/mol, respectively, indicating efficient binding. The results showed that the

299

affinity of Z100 was stronger than that of Z93, as determined by comparing the numbers

300

of non-covalent bonds (π-π interactions, hydrogen bonds, and hydrophobic interactions). It

301

has been established that the results of computational analyses often correlate well with the

302

outcomes of experimental studies 42. The A and U nucleotides of Z100 mainly interacted,

303

suggesting that the A and U nucleotides were key for binding, which supplied possible

304

binding sites of A=T in the ZEN aptamer 27. On the other hand, the results of replacements

305

docking between G, C and A, U in U-71, U-72, A-38, U-39, A-69 and U-35 of Z100, were

306

showed in Figure S3. Four replacement strategies, (a) G, C substituted for A, U, (b) C, G

307

substituted for A, U, (c) G substituted for A, U, and (d) C substituted for A, U, were showed

308

lower binding free energies [(a) -8.7, (b) -8.9, (c) -8.5 and (d) -8.1 kcal/mol] compared with

309

Z100 (-9.6 kcal/mol), respectively. The binding sites of ZEN were mainly located on 40 nt

310

random sequences of Z100 (Figure 4c). On the contrary, the binding sites of ZEN were

311

mainly located on 10 nt random sequences of Z93, suggesting that the random sequences

312

were principal binding sites and the designs of the aptamers were both rational and



313

effective. Otherwise, the docking results were aligned according to affinity, which pointed

314

to the correct binding mode as the energetically most favorable and also suggested that the

315

AutoDock score function was effective for qualitative evaluation of the ligand-DNA

316

interactions 34.

317 318

Figure 4 Characterization of aptamers. (a) The calculated Kd values of Z100 and Z93 were


Page 18 of 33

Page 19 of 33


319

in the high nanomolar range (15.2 ± 3.4 and 440.0 ± 129.5 nM, respectively). The 2-D (b)

320

structures of the aptamers were predicted using the mfold and 3dRNA-V2.0 online tools.

321

(c) molecular docking study was performed to investigate the binding mode between the

322

ZEN molecule and aptamers. The best-scoring pose as judged by the Vina docking score

323

was chosen.

324

3.3 Detection of ZEN with a label-free detection method based on the selected aptamer

325

The concentration of the AuNPs was about 14 nM and the particles had uniform diameters

326

of 13 nm (Figure 5). The dispersity and proportion of the nanoparticles were acceptable,

327

and the morphology was elaborately controlled. AuNPs are widely used for the

328

establishment of visual mycotoxin label-free aptasensors 24. ssDNA was absorbed on the

329

surface of the AuNPs, which protected the AuNPs (red) against salt-induced aggregation

330

(blue) 29. The optimum protective dose of Z100 was confirmed at 25 μL and 0.25 μM.

331 332

Figure 5. Transmission electron microscope studies of synthesized AuNPs, bar = 100 nm

333

Different concentrations of Z100 (0.125, 0.25, 0.5 and 1 μM) were mixed with ZEN



334

(0, 4, 8, 16, 32, 64 and 128 ng/mL). The spectra and color changes are shown in Figure 6a

335

and b revealed that the sensitivity was reduced as the amount of the aptamer decreased.

336

The absorption spectra at 520 nm showed a gradual decrease as ZEN concentrations

337

increased from 0.25 μM to 1 μM aptamer. The Z100 aptamer at 0.25 μM was the most

338

suitable working concentration because of visible color changes with aggregated AuNPs in

339

the presence of ZEN at different concentrations (Figure 6a and b). The absorbance at 520

340

nm was plotted to the LN [ZEN concentrations] (Figure 6c) to identify correlations between

341

absorbance and ZEN concentrations. Excellent linearity was shown by linear range of 4-

342

128 ng/mL (12.5- 402.1 nM) with a detection limit of 4 ng/mL (12.5 nM). The correlation

343

coefficient (R2) of the standard curve was 0.9783 (Figure 6b and c). Our detection limits

344

were lower than the regulation of European commission (2006/576/EC) 3, European

345

commission (1881/2006) 4, Canadian Food Inspection Agency 5, US Food and Drug

346

Administration 6, and the People’s Republic of China (GB 13078-2017) 7. As one of the

347

most heavily contaminations from 2013 to 2017 in China 43-47, the average value of ZEN

348

was 96.06 μg/kg in 6797 feed and grain samples across the country, with average positive

349

detective rate of 82.27 %, which were under our detection ranges, and proved the

350

practicality of this method.

351

By contrast, α-ZON, β-ZON, AFB1, T-2 and FB1 were introduced to the label-free

352

ZEN detection system using the Z100 aptamer at a concentration of 0.25 μM. The results

353

indicated that the negative control of structural analogue group was effective in detection

354

system (Figure 6b).


Page 20 of 33

Page 21 of 33


355 356

Figure 6 The sensitivity for ZEN detection depending on aptamer amount adsorbed onto

357

AuNPs at OD520. (a) Absorption spectra of AuNPs solution at various amounts of aptamer.

358

(b) Corresponding photographic images. (c) Typical calibration curves for ZEN with

359

various amounts of aptamer adsorbed to AuNPs.

360

3.4 Recovery Rates of the ZEN from real samples

361

The developed label-free aptasensors were further tested for ZEN-free sample analysis in

362

practical applications. As shown in Table 3, good recovery percentages were obtained at



Page 22 of 33

363

range from 96.42% to 99.78% of corn powder and 95.99% to 103.73% of feed by typical

364

calibration curves, and relative standard deviation (RSD) was within 7.63%, which

365

suggested that the simpler the composition of the samples, the better the recovery values.

366

Table 3 Recoveries of ZENs for applicability of the AuNPs based aptasensor assay in real

367

samples of spiking study (n=3) Real samples of

Spiked levels

Detected concentration

Recovery

spiking study

(μg/kg)

(μg/kg)

ratio (%)

corn powder

16

15.43

96.44

1.07

32

31.25

97.66

1.91

128

127.72

99.78

7.13

16

15.36

95.99

1.17

32

31.30

97.81

2.80

128

132.78

103.73

7.63

RSD (%)

feed of mouse

368

Each on-site sample was detected by Huaan Magnech ELISA kits (Table 4). The sample

369

with lowest ZEN concentration (sample B of 50.82± 8.58 μg/kg) was used to prepare

370

typical calibration curves of on-site samples for applicability of the AuNPs-based

371

aptasensor assay. After the addition of target concentrations of ZEN (80, 160, 320, 640 and

372

1320 μg/kg), the standard concentrations of sample B were 79.59± 1.24, 159.14± 9.28,

373

318.86± 6.99, 654.15± 29.48 and 1302.62± 39.25 μg/kg. A representative calibration curve

374

for the AuNPs-based aptasensor assay is presented in Figure 7. Each on-site sample showed


Page 23 of 33


375

good recovery percentages in the range of 94.31% to 105.87% and RSD within 19.14%.

376

The detection limit was lower than 50 μg/kg. The ZEN concentrations of the samples of

377

contaminated feed exceeded the detection range (1.75- 4.05 μg/kg) of the R-Biopharm kit

378

(results not shown), indicating that the R-Biopharm kit was suitable for precise laboratory

379

analysis. The results revealed successful application of the selected aptamers and the

380

developed aptasensors were sufficient to screen for ZEN residues in actual samples. 0.35

Absorbance

0.30 0.25 0.20 0.15 0.10 4

5

381

6 LN[ZEN concentration]

7

8

382

Figure 7 Typical calibration curves for ZEN with 0.25 μM aptamer adsorbed at OD520 to

383

AuNPs in on-site contaminated feeds (B samples of condensed feeds of lactation cow).

384

Table 4 Recoveries of ZEN for applicability of the AuNPs based aptasensor assay in real

385

samples of on-site contaminated feeds (n=3) Real samples of

Detected Sample

ELISA kits

on-site

RSD

ratio (%)

(%)

105.87

2.69

concentration numbers

(μg/kg)

contaminated feeds condensed feeds of

Recovery

(μg/kg) A

71.02

75.19



lactation cow

B C

complete feed of

D

pregnancy sow

E F

complete feed of

G

layer

H I

Page 24 of 33

50.82

51.08

100.50

3.96

89.29

89.16

99.86

3.39

270.57

255.08

94.31

10.07

193.62

202.67

104.68

16.57

331.21

337.51

101.90

19.14

146.15

142.14

97.26

13.44

119.51

120.11

100.50

14.36

197.23

189.89

96.28

8.43

386

In conclusion, an ssDNA aptamer with high binding affinity and specificity to ZEN

387

was developed using an immobilization-free selection method. The molecular simulations

388

offered key insights and rational explanations of the interactions between ZEN and the

389

aptamers. On the basis of the observed color change of AuNPs, a label-free method for

390

detection of ZEN was successfully developed. The linear dynamic range and detection

391

sensitivity were 12.5-402.1 nM and 12.5 nM, respectively. These results show the potential

392

of the aptamer and the reliability of the method for the rapid detection of ZEN, and provide

393

valuable information for further development of aptamer inhibitors to ensure the safety of

394

animal feed.

395 396

Conflicts of Interest

397

The authors declare that they have no conlicts of interest in the present study. The authors


Page 25 of 33

398


declare no competing financial interest

399 400

Acknowledgments

401

This study was financially supported by the National Natural Science Foundation of China

402

(31601974 and 31700140) and Shanghai Science and Technology Commission Research

403

Program （ 16140900500 ） . Supporting Information Available: [Annealing temperature

404

optimization of amplification; The Sequences of 13 aptamers; The specificity of 13

405

aptamers; Replacements docking between G, C and A, U. This material is available free of

406

charge via the Internet at http://pubs.acs.org.]



407

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Selection of a DNA Aptamer against Zearalenone and Docking

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