Quantum-Dot-Based Lateral Flow Immunoassay for Detection of

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Quantum dot-based lateral flow immunoassay for detection of neonicotinoid residues in tea leaves Shuangjie Wang, Ying Liu, Shasha Jiao, Ying Zhao, Yirong Guo, Mengcen Wang, and Guonian Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03981 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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Journal of Agricultural and Food Chemistry

1 2 3

Quantum dot-based lateral flow immunoassay for detection of neonicotinoid residues in tea leaves

4 5

Shuangjie Wang1, Ying Liu1, Shasha Jiao1, Ying Zhao1, Yirong Guo1,

6

Mengcen Wang1†, Guonian Zhu1

7 8

1

Institute of Pesticide and Environmental Toxicology, Zhejiang

University, 310058 Hangzhou, China

9 10

†Address for Correspondence:

11

Mengcen Wang, Ph.D.

12

Institute of Pesticide and Environmental Toxicology, Zhejiang University

13

E-mail: [email protected]

14

Phone & Fax: +86-571-88982517

1

ACS Paragon Plus Environment


15

Abstract

16

Neonicotinoid insecticides are commonly used for pest control on tea

17

plantations due to their broad-spectrum activity. However, neonicotinoid

18

residues released from tea leaves into tea infusions pose a dietary risk to

19

consumers. Therefore, a rapid, sensitive and reliable on-site detection

20

method for neonicotinoids is needed. We developed a quantum dot-based

21

fluorescent lateral flow immunochromatographic strip (LFICS) combined

22

with a broad-specific antibody for detection of typical neonicotinoids

23

(imidacloprid, imidaclothiz, and clothianidin), with sensitivities (IC50, 50%

24

inhibitory concentration) of 0.104–0.33 ng/mL and visual detection limits

25

of 0.5–1 ng/mL. The strip assay could be completed in less than 30

26

minutes. Using the LFICS to analyze spiked tea samples (green tea, black

27

tea, and oolong tea), the average recovery of the three neonicotinoids

28

ranged between 71% and 111%, with coefficients of variation below 12%.

29

The results from the LFICS tests for field samples were consistent with

30

results from ultra-performance liquid chromatography-tandem mass

31

spectrometry. The newly-developed strip is a useful tool for the on-site

32

detection of neonicotinoid residues in tea.

33 34

Keywords: lateral flow immunoassay; quantum dot; neonicotinoids; tea

35 36 2


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37

Introduction

38

Teas can be classified into three types (green tea, oolong tea and

39

black tea) based on fermentation processing1. Tea plants are attacked by

40

many sucking insects, and insecticides are commonly used for their

41

control. Among the insecticides used, neonicotinoids are effective for

42

controlling whiteflies, aphids, and leaf hoppers. Neonicotinoids have the

43

advantages of a broad host spectrum, long residual activity and unique

44

modes of action. They have become the most widely used insecticides in

45

the world 2. Neonicotinoids are relatively polar compounds and are easily

46

leached from dry tea or the surface of treated tea into drinkable tea

47

infusions. This creates a risk of human exposure to these pesticide

48

residues 3. Hence, it is necessary to monitor neonicotinoid residues in teas

49

to increase tea quality and safety.

50

Various instrumental analytical approaches have been used to detect

51

neonicotinoids. These include high-performance liquid chromatography

52

coupled

53

chromatography tandem mass spectrometry (LC-MS/MS)5, and ultra

54

performance

55

(UPLC-MS/MS)6, 7. All of these methods are acceptably sensitive,

56

accurate and selective, but the processes are complex, and the necessary

57

equipment is expensive. Therefore, a portable, sensitive, rapid and

58

easy-to-use method, which can also be used outside the laboratory, was

with

a

liquid

diode

array

detector

(HPLC-DAD)4,

chromatography-tandem

3


mass

liquid

spectrometry


59

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developed for neonicotinoid detection.

60

Immunoassays, based on antibody-antigen interactions, have been

61

widely applied in disease diagnosis and biochemistry. Immunoassays

62

benefit from the specific recognition of antigen by an antibody, which

63

reduces the onerous procedures of sample pretreatment. Enzyme-linked

64

immunosorbent assay (ELISA) is a common immunoassay for residue

65

analyses of neonicotinoids

66

and cannot be used for rapid on-site screening. In contrast, one-step

67

lateral flow immunochromatographic strips (LFICSs) can reduce some of

68

the ELISA difficulties 10.

69

8, 9

. However, ELISA requires multiple steps

Colored nanoparticles and luminescence materials have been used as 11,

12

70

detection probes in LFICS

. Among these, colloidal gold,

71

characterized by its tunable optical properties and stability under liquid

72

and dry conditions, is a common probe used in LFICS. Several studies

73

have used nanogold-based LFICS for the detection of neonicotinoids. A

74

nanogold-based immunostrip was developed for the simultaneous

75

detection of imidacloprid and thiamethoxam, and the visual detection

76

limits in the assay buffer were 0.5 and 2 ng/mL, respectively

77

nanogold-based signal amplified immunochromatographic assay was

78

developed for semi-quantitative detection of imidacloprid

79

studies describing the use of other nanoparticle-labelled strips for the

80

detection of neonicotinoids have not been reported. 4


13

. A

14

. However,

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81

Quantum dot (QD) is a new type of fluorescent nanoparticle

82

semiconductor material. It is characterized by its broad adsorption,

83

narrow photoluminescence spectra, size-tunable emission, strong

84

luminescence and high photostability

85

functional groups, such as carboxyl, to achieve water solubility and

86

biocompatibility. Their unique properties make them suitable for a wide

87

array of biotechnological and bio-analytical studies

88

LFICS consumed less immunoreagents and was more sensitive than the

89

colloidal gold-based LFICS

90

immunostrips. Thus far, QDs have been used as probes in

91

microwell-based fluorescent-linked immunosorbent assays for the

92

detection of neonicotinoids such as clothianidin and thiacloprid

93

imidaclothiz

94

detection, and no studies of QD-based LFICS for neonicotinoid detection

95

have been published. In this study, we used QD as the label to develop a

96

rapid, sensitive, portable fluorescent LFICS for detecting three

97

neonicotinoids (imidacloprid, imidaclothiz, and clothianidin) in tea

98

samples.

23

15

. QDs can conjugate with

16-18

. The QD-based

19-21

. Therefore, QD is a promising label for

22

and

. There are few reports of QD-based LFICS for pesticide

99 100

Material and methods

101

Reagents and materials

102

Standards of eight neonicotinoids (imidacloprid (99.0%), dinotefuran 5



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103

(97.5%), nitenpyram (99.0%), acetamiprid (99.0%), imidaclothiz (99.0%),

104

thiacloprid (98.0%), thiamethoxam (99.0%), clothianidin (99.5%), were

105

purchased from Dr. Ehrenstorfer (Augsburg, Germany). The carboxylic

106

group-modified CdSe/ZnS core-shell QDs (emission at 605 ± 5 nm) were

107

provided by Jiayuan Quantum Dots Co., Ltd. (Wuhan, China). A

108

broadly-specific monoclonal antibody (mAb) against imidacloprid and its

109

analogues was previously prepared in our laboratory, as well as the

110

corresponding coating antigen. The 1-ethyl-3-(3-dimethylaminopropyl)

111

carbodiimide hydrochloride (EDC) Tween-20, bovine serum albumin

112

(BSA), polyvinyl pyrrolidone (PVP) and sucrose were provided by

113

Aladdin

114

secondary amine, PSA) was obtained from Agela Technologies (Tianjin,

115

China). Polyvinyl polypyrrolidone (PVPP) was obtained from Sigma

116

(Steinheim, Germany). All other inorganic chemicals and organic

117

solvents were of analytical reagent grade or better. Purified water was

118

obtained using a Milli-Q water purification system (Millipore, Bedford,

119

MA, USA). Glass-fiber membrane CFCP203000 was used for loading

120

conjugate and the absorption membrane CFSP223000 were from

121

Millipore. The nitrocellulose (NC) membranes were purchased from

122

different companies, including Sartorius-CN-140 (Gottingen, Germany),

123

Millipore HiFlow-135 and HiFlow-180 (Billerica, USA).

124

Preparation of QD-mAb conjugates

(Shanghai,

China).

N-propyl-ethylenediamine

6


(primary

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125

The conjugation strategy for the preparation of QD-based antibody

126

was modified from a previous report 22. A quantity of carboxyl-modified

127

QDs (25 µL, 8 µM in 50 mM borate buffer, pH 9.0) was mixed with

128

borate buffer (10 mM, pH 8.0) under magnetic stirring. Then, the

129

antibodies (1.64 – 24.5 µL, 9.17 mg/mL) and EDC (7.7 µL, 10 mg/mL)

130

were added into the previous solution. The mixture (200 µL) was

131

incubated for 120 min at 4 °C in darkness with stirring at 320 rpm. This

132

was followed by centrifugation (5000 rpm, 10 min) by a 5-mL

133

ultra-filtration concentrator (MWCO 3K, Millipore). The supernatant was

134

then removed and the conjugate was resuspended with borate buffer (10

135

mM, pH 7.5) containing 1% BSA, 0.05% PVP and 1% sucrose. The final

136

conjugate solution was stored at 4°C.

137

Assemblage of the one-step strips

138

A one-sided adhesive polyvinyl chloride (PVC) sheet was used as a

139

support for the strip composition. The absorption membrane and the glass

140

fiber membrane were pasted on the sheet, overcrossing 2 mm with the

141

two ends of the NC membrane. These composites were stored in a

142

desiccator at 4 °C before use.

143

Preparation of QD-based LFICS

144

Coating antigen and goat anti-mouse antibody were immobilized

145

onto the NC membrane as the test and control lines, respectively. The

146

membrane was dried at 37°C for 2 hr. The obtained composite was cut 7



147

into individual strips and stored in a desiccator at 4°C before use.

148

Strip assays for imidacloprid

149

The QD-mAb conjugate was diluted (400X) by borate buffer before

150

using. Standard or sample solution (25 µL) and the QD-mAb conjugate

151

(25 µL) were mixed and added to wells of the 96-well microtiter plate.

152

Then, the strip was inserted into the well of 96-well plate. After 25 min,

153

the visible signal was observed under 365nm UV excitation and the

154

fluorescence intensity was recorded by a fluorescent reader (365 nm

155

excitation, 610 nm emission), as shown in Fig. 1.

156

To offset the heterogeneity of the strips, the fluorescence intensity

157

ratio of T-line to C-line (T/C) was used for quantitative analysis and this

158

minimized environmental factors potentially affecting fluorescence

159

intensity. Standard curves were obtained by plotting the fluorescence

160

intensity ratio of the T-line to the C-line (T/C) (as Y-axis) against the

161

analyte concentration (X), and they were fitted into a four-parameter

162

logarithmic equation.

163

Y = A2 + [(A1- A2) / 1 + (X / X0)p], where A1 is the maximum value of

164

T/C in the logarithmic equation, while A2 is the minimum value; X0 is

165

equal to IC50 (50% inhibitory concentration), taken as the assay sensitivity,

166

and p indicates the slope of the curve at IC50. The linear working range

167

was represented by IC20–IC80.

168

Cross-reactivity 8


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Cross-reactivity (CR) was used to express the selectivity of the strip

170

assay.

171

neonicotinoids (dinotefuran, nitenpyram, acetamiprid, imidaclothiz,

172

thiacloprid, thiamethoxam, and clothianidin) were all tested by the strips.

173

CR values were calculated as follows:

174 175

The

standard

solutions

of imidacloprid

and

the

other

CR % = (IC50 of imidacloprid / IC50 of the other neonicotinoids) × 100 Tea matrix effect on LFICS

176

Neonicotinoids have good water solubility and tea is commonly

177

brewed with boiling water. Thus, each sample (1 g) of tea was extracted

178

with 10 mL of boiling water. After 30 min, the extracted tea infusions

179

were diluted with different volumes of borate buffer. Matrix effects were

180

determined by comparing standard curves in the matrix extracts with the

181

curve prepared using matrix-free borate buffer.

182

Recovery tests for tea samples

183 184

Neonicotinoid-free tea samples confirmed by UPLC-MS/MS were used as blank samples for recovery tests.

185

Blank tea samples (1 g of dried black tea, dried green tea, or oolong

186

tea) were spiked with imidacloprid at 0.04-320 mg/kg and left standing

187

for 30 min. Then, tea samples were extracted with 10 mL of boiling water

188

for 30 min. The supernatant was then diluted with borate buffer (10 mM,

189

pH 7.5) and used for LFICS analysis. Each analysis was performed in

190

four replicates and continued for 4 d. Then, the recovery (the calculation 9



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191

formula listed in the supporting information) and coefficient of variability

192

(CV) were calculated.

193

Analysis of authentic samples

194

Tea samples (2.0 g) collected from different production areas (S1-S8)

195

were placed into 50-mL centrifuge tubes and 10 mL of boiling water was

196

added. The samples were incubated for 30 min. Two methods were used

197

to study neonicotinoid levels in the samples. For the strip test, the

198

supernatant was diluted with borate buffer (10 mM, pH 7.5) and then it

199

was ready for LFICS analysis. UPLC-MS/MS was also used for testing.

200

The sample-pretreatment method was modified from previous studies

201

3, 6,

24

. The incubated solution was extracted by vigorous shaking for 30 min

202

with acetonitrile (20 mL). Then, NaCl (5 g) and MgSO4 (5 g) were added

203

and the mixture was vigorously shaken for 1 min. Then, the mixture was

204

centrifuged (6000 rpm, 8 min) and the supernatant was transferred to a

205

100-mL flat-bottomed flask. The extraction was then concentrated using a

206

rotatory evaporator and dried by nitrogen gas at 40 °C. The residue was

207

dissolved in 2 mL of methanol. PSA (0.1 g) and PVPP (0.3 g) were added

208

to the residue solution, followed by vortexing for 2 min, and

209

centrifugation (6000 rpm, 5 min). The supernatant was filtrated through

210

microporous film (0.22 µm) before undergoing UPLC-MS/MS.

211

UPLC-MS/MS analysis and validation

212

The authentic samples were analyzed by UPLC-MS/MS. The UPLC 10


Page 11 of 34


213

system consisted of an Acquity ultra-performance liquid chromatograph

214

(Waters, Milford, MA). Chromatographic separations of neonicotinoids

215

were performed on a UPLC HSS C18 SB Column (1.8 µm, 2.1 × 100 mm

216

i.d. Acquity). The mobile phase consisted of 60% solvent A (0.1% of

217

formic acid in water, v/v) and 40% solvent B (acetonitrile). A subsequent

218

equilibration time (10 min) was performed before injection. The flow rate

219

was 0.3 mL/min, the injection volume was 10 µL, and the column and

220

sample temperatures were maintained at 40 °C and 8 °C, respectively.

221

The MS/MS analysis was performed by Applied Biosystems Triple

222

Quad 5500 (Foster City, CA, USA) in electrospray positive-ion multiple

223

reaction modes. The parameters of m/z and collision energy of precursor

224

ions and quantitative product ions from neonicotinoids

225

Table S1. Source–dependent parameters were as follows: ion spray

226

voltage, 5500 V; curtain gas, 20 psi; ion source temperature, 200 °C;

227

atomization air pressure, 20 psi; auxiliary gas, 20 psi; collision-activated

228

dissociation, 4 V. The AB Sciex Analyst 1.6 software (Applied

229

Biosystems) was used for data acquisition and evaluation.

24

are shown in

230 231

Results and discussion

232

The lateral flow immunoassay was based on a competitive format

233

(Scheme 1). The target pesticide in the samples and the antigen coated on

234

the test line compete for binding to the antibody conjugated with QDs. In 11



235

the absence of pesticides in the sample, some of the Ab-QDs conjugates

236

will bind to the antigen on the test line, and the remaining conjugates will

237

bind to goat anti-mouse antibody on the control line, expressed by the

238

equalization of the fluorescence intensity between the T-line and C-line.

239

In the case of excess pesticides in a sample, the Ab-QDs combined with

240

the pesticides, and there were no Ab-QDs conjugates able to bind to the

241

T-line; the fluorescence intensity of the T-line therefore decreased.

242

Molar ratio of quantum dot to antibody

243 244

Under UV 365 nm excitation, the maximum emission wavelength of the QDs was displayed (Fig. S1).

245

Theoretically, the free antigen in the sample solution should

246

completely occupy the binding site of antibody coupled with QDs, so that

247

the antigen immobilized on the T-line could not bind with the conjugate

248

again. However, there was often excessive coupling-antibody, which led

249

to the QD-Ab conjugate’s binding with both the free analyte and the

250

immobilized antigen. This phenomenon would decrease the sensitivity.

251

Therefore, it is important to optimize the molar ratio of the QD to the

252

antibody.

253

Foubert proposed that each QD can conjugate with 2–10

254

immunoglobulins 25. Hence, the effect of different molar ratios of QD to

255

the mAb (1:1, 1:5, 1:10, and 1:15) on the assay’s performance was tested.

256

Among these, the 1:1 group gave no fluorescence signal, and the 1:10 and 12


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257

1:15 groups had lower sensitivities (Fig. 2). Thus, the 1:5 group was the

258

optimal molar ratio. The results were consistent with a previous study

259

showing that a higher molar ratio of antibody (IgG) to QD could decrease

260

assay sensitivity because it was difficult to completely block antibodies

261

by the analytes 18.

262

Optimization of experimental parameters

263

The analytical performance of LFICS can be affected by the

264

properties of the materials used to fabricate the device, particularly the

265

working membrane. Three types of NC membranes frequently used in

266

LFICS, including Millipore HiFlow-180, Millipore HiFlow-135, and

267

Satorius CN-140, were used to evaluate the sensitivity and fluorescent

268

intensity of LFICSs. All tested membranes showed an equilibrium of

269

intensity between the T-line and C-line. Sartorius CN-140 achieved the

270

desired fluorescent intensity and sensitivity (IC50) and was determined to

271

be the optimal nitrocellulose membrane (Fig. 3).

272

The concentrations of the coating antigen (0.1 to 1 mg/mL) in the test

273

line and goat anti-mouse antibody (0.01 to 0.5 mg/mL) in the control line

274

were adjusted using a matrix approach. If the T-line intensity is much

275

stronger than that of C-line, it will be judged as a false-negative and

276

reduce the sensitivity. On the contrary, it will be judged as a false-positive.

277

We found that 1 mg/mL of the coating antigen and 0.025 mg/mL of the

278

secondary antibody provided the optimal working concentrations. 13



Page 14 of 34

279

To prevent the non-specific binding in the assay, BSA was used to

280

block the leftover spaces over the solid surface after immobilization of a

281

captured biomolecule. Based on the additives used in previous studies

282

26,

27

, the glass-fiber was pretreated with PBST containing 0.25% BSA, 0.25%

283

PVP and 5% sucrose to provide good dispersion of coupling-QDs during

284

the strip tests.

285

To establish the optimal reaction system for good sensitivity and

286

good signal intensity for both the T-line and C-line, 10 mM borate buffer

287

at different pH levels of 7.0, 7.5, 8.0, and 8.5 were evaluated. There was

288

no obvious difference in sensitivity and T /C among these groups (Fig. 4).

289

Similar to other reports

290

condition.

291

Determination of imidacloprid by LFICS

20, 22, 28

, pH 7.5 was chosen as the working

292

Under the optimal conditions, a series of known concentrations

293

(0.024 to 100 ng/mL) of imidacloprid were prepared with borate buffer

294

(10 mM, pH 7.5) to produce a standard calibration curve (Fig. 5a). With

295

increasing imidacloprid concentration, the fluorescence intensity of the

296

T-line gradually decreased (Fig. 5b). Quantitative measurement was

297

further conducted by the fluorescent strip reader. Results showed that the

298

IC50 of LFICS for the detection of imidacloprid was 0.104 ng/mL, and the

299

linear range was 0.012–0.88 ng/mL (IC20–IC80). For semi-quantitative

300

visual detection, we set the cutoff value (visual LOD) at 0.5 ng/mL for 14


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301

imidacloprid, at which concentration the signal intensity of the T-line was

302

faint (Fig. 5c).

303

Selectivity of LFICS

304

The selectivity of the test strip was evaluated by testing the cross

305

reactivity (CR) of the assay with seven other neonicotinoid insecticides

306

(dinotefuran,

307

thiamethoxam, and clothianidin). The LFICS had a high degree of

308

cross-reactivity with imidaclothiz (61.2%) and clothianidin (31.5%),

309

whereas it displayed negligible cross-reactions to the other five

310

neonicotinoids (CR < 1.5%) (Table 1). This indicated that the strips could

311

also be used to detect imidaclothiz (IC20 to IC80: 0.028 to 1.13 ng/mL,

312

limit of visual detection: 0.5 ng/mL) and clothianidin (IC20 to IC80: 0.039

313

to 2.90 ng/mL, limit of visual detection: 1 ng/mL) with good sensitivities.

314

For imidaclothiz and clothianidin, the calibration curves and photos under

315

365 nm excited UV light are shown in Fig. S2 and Fig. S3. Using the strip

316

reader

317

neonicotinoids were higher than those from most studies (Table 2).

318

Tea matrix effects on LFICS

for

nitenpyram,

quantification,

acetamiprid,

the

imidaclothiz,

assay sensitivities

to

thiacloprid,

the

three

319

Sample pretreatment prior to analysis by UPLC-MS/MS was tedious.

320

A rapid, simple and on-site sample-pretreatment method with a

321

high-extraction rate was needed. We diluted the tea infusion with assay

322

buffer to reduce the matrix effect. Results shown in Fig. 6 indicate that 15



323

different tea matrices can have different influences on assay performance,

324

which was consistent with the findings of Jiao et al.6. Excess dilution can

325

reduce the assay’s sensitivity. A 160-fold dilution of green tea, 80-fold

326

dilution of black tea, and 20-fold dilution of oolong tea were selected for

327

LFICS to produce a negligible matrix effect.

328

Considering the dilution factors for matrix effects, the visual LOD of

329

imidacloprid and imidaclothiz were 0.01–0.08 mg/kg in teas and that of

330

clothianidin was 0.02–0.16 mg/kg in tea. Thus, the newly-developed

331

LFICS had a desirable naked-eye sensitivity that satisfied the

332

requirements of the maximum residue limits (MRLs) for imidacloprid

333

(0.5 mg/kg) and imidaclothiz (3 mg/kg) by the National Food Safety

334

Standard of China (GB2763-2016) and for clothianidin (0.7 mg/kg) by

335

the European Union (EU). Additionally, with the aid of the strip reader,

336

the LOD of imidacloprid in tea was 8 × 10-6–6.4 × 10-4 mg/kg, which

337

could satisfy the EU MRL requirement for imidacloprid (0.05 mg/kg).

338 339

Analysis of spiked samples by LFICS

340

Data showing the accuracy and precision of spiked samples are

341

presented in Tables 3, S2, and S3. The spiked levels were selected to be

342

between the assay working range and naked-eye sensitivity. Acceptable

343

recovery of 70.71%–110.78% was obtained, with inter-day CVs of

344

6.89%–11.67% and intra-day CVs of 1.94%–12.49%. The LFICS would 16


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345

be suitable for the rapid detection of imidacloprid, imidaclothiz, and

346

clothianidin in green tea, black tea and oolong tea, with desirable

347

sensitivities meeting the MRL requirements.

348

Analysis of authentic samples

349

To further evaluate the reliability of the strip test, eight tea samples

350

(S1–S8) were collected from different tea-producing areas, detected by

351

LFICS, and then confirmed by UPLC-MS/MS.

352

Since the broadly-specific antibody was unable to completely

353

distinguish imidaclothiz, imidacloprid and clothianidin from each other, a

354

standard calibration curve of three mixed pesticides was established to

355

determine the total amount of the three pesticides in the samples. The

356

mixture curve was in accordance with the individual curves of

357

imidacloprid, imidaclothiz, and clothianidin (Fig. S5) when the total

358

concentration of neonicotinoids was in the detection level. Therefore, the

359

mixture curve was used to calculate the unknown concentration of

360

neonicotinoids in the tea samples. As shown in Table 4, 3 out of the 8

361

samples were positive, with concentrations of 0.03–0.125 mg/kg. With

362

the help of UPLC-MS/MS confirmation, the exact types of pesticides

363

were identified (Table 4). The results detected by the LFICS were

364

consistent with those from UPLC-MS/MS.

365

We developed an LFICS employing quantum dots as fluorescent

366

probes for the rapid detection of neonicotinoid insecticides. The major 17



Page 18 of 34

367

ratio of quantum dot to antibody, the pH of assay buffer, sensitivity,

368

selectivity, matrix effects, and the assay’s accuracy and reliability were

369

investigated. Using the strip reader, we found that the sensitivities of the

370

QD-based LFICS for the detection of three neonicotinoids (imidacloprid,

371

imidaclothiz, and clothianidin) were higher than those of previously

372

reported

373

neonicotinoids can fully meet their MRLs on teas in China and partly

374

reach the EU MRLs.

immunoassays.

Moreover,

the

visual

LOD

of

three

375

LFICS can simultaneously detect three neonicotinoids, which was

376

convenient for screening pesticides commonly used for insect control on

377

tea plants. The rapid, sensitive and portable QD-based LFICS could be

378

applied to screen neonicotinoid residues by the naked eye on-site or under

379

outside laboratory conditions. This will contribute to the regulation of

380

neonicotinoid use on tea and other agricultural products and reduce the

381

risk of human exposure to neonicotinoid residues.

382 383

Acknowledgements

384

This research was financially supported by National Key R&D

385

Program of China (2017YFF0210200), National Natural Science

386

Foundation of China (31401768) and the Agricultural Project for Public

387

Technology Research in Zhejiang province (2016C32004).

388 389

Conflict of interests 18


Page 19 of 34


390 391 392

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Page 24 of 34

Page 25 of 34


522

Figures

523

524 525

Fig. 1. The display of quantum-dot (QD)-labelled lateral flow

526

immunochromatographic strip (LFICS) under UV excitation and placed

527

in a fluorescent reader.

528

16 14

IC50 (ng/mL)

12

T/C in Control group

18

10

4

2

0 1:5

1:10

1:15

Molar Ratio

8 6 4 2 0 1:5

1:10

1:15

Molar Ratio

529 530

Fig. 2. Effects of the molar ratio of quantum dots to imidacloprid

531

antibody (1:5, 1:10, 1:15) on the assay performance. Bar, ± SD (n=4)

532 25



5

T/C IC50

4

4

3

3

2

2

1

1

0

IC50 (ng/mL)

T-line to C-line Ratio

5

Page 26 of 34

0 Millipore HiFlow-135

Satorius CN-140

Millipore HiFlow-180

Working Membrane

533 534

Fig. 3. The sensitivity (IC50) and T/C ratio of strips based on different

535

working membranes. Bar, ± SD (n=4)

536

1.5

1.5

1.0

1.0

0.5

0.5

0.0

IC50 ( ng / mL )


T/C IC50

0.0 pH 7.0

pH 7.5

pH 8.0

pH 8.5

Levels of pH in Working buffer

537 538

Fig. 4. Effects of pH levels (pH 7.0, 7.5, 8.0, 8.5) in working buffer on

539

the assay performance. Bar, ± SD (n=4)

540

26


Page 27 of 34


T/C


1.0

0.5

0.0 1E-3

0.01

0.1

1

10

100

Concentration of Imidacloprid (ng/mL)

541

（a）

542

543

(b)

544

(c)

545

Fig. 5. (a) The calibration curve of imidacloprid in borate buffer (10 mM,

546

pH 7.5). Bar, ± SD (n=4) (b) The photo of test strips with different

547

concentrations (0.003 - 100 ng/mL) of imidacloprid under 365 nm UV

548

excitation. (c) Test strips treated with borate buffer (imidacloprid-free)

549

and borate buffer containing imidacloprid (0.5 ng/mL) under 365 nm UV

550

excitation. 27



Green Tea

1.0

0.5

1.0

0.5

1E-3

0.01

0.1

1

10


100

BB Buffer 10 - fold 20 - fold 40 - fold 80 - fold 160 - fold

1.0

0.5

0.0

0.0

0.0

1.5


1.5

Oolong Tea BB Buffer 10 - fold 20 - fold 40 - fold 80 - fold 160 - fold

1.5



Black Tea BB Buffer 10 - fold 20 - fold 40 - fold 80 - fold 160 - fold

2.0

Page 28 of 34

1E-3

0.01

0.1

1

10


100

1E-3

0.01

Fig. 6. Matrix effects of different tea types on the assay performance. Bar, ±SD (n=4)

28


0.1

1

10


100

Page 29 of 34


Tables

Table 1. The cross reactivity (CR) of the strip test to eight neonicotinoids. Analytes

IC50 (ng/mL)

CR(%)

0.104

100

Imidaclothiz

0.17

61.2

Clothianidin

0.33

31.5

Thiacloprid

7.885

1.31

Nitenpyram

8.701

1.2

Acetamiprid

14.101

0.7

Dinotefuran

>1000

1000

5000 (IC50)

Colloidal gold-based strips

Imidacloprid

0.5 (visual LOD)

Signal amplified colloidal gold-based strips Quantum dot-based ELISA

Imidacloprid

10 (visual LOD)

Tomato, cabbage, rice Tomato, cucumber, apple Pond water, rice field water, canal water, fish pond, Dushu lake water Cucumber, tomato, lettuce, apple, orange Chinese cabbage

Clothianidin

12.5 (IC50)

22

Quantum dot-based strip

Imidacloprid

0.104, 0.5 (IC50, visual LOD) 0.17, 0.5 (IC50, visual LOD) 0.33, 1 (IC50, visual LOD)

Water, soil, cabbage, rice, tomato Green tea, black tea, oolong tea

Imidaclothiz Clothianidin

30


9

30

31

13

14

This article

Page 31 of 34


Table 3. The accuracy and precision of the developed LFICS for the detection of imidacloprid in teas a. Tea types

Spiked level (ng/kg)

Green Tea

Black Tea

Oolong Tea

a

3.2 80 128 1.6 40 64 0.4 10 16

Intra-batch (n=4)

Inter-batch (n=4)

Mean ± SD (ng/g)

CV (%)

Recovery (%)

2.68±0.29 72.94±7.64 119.99±12.21 1.42±0.12 39.62±4.62 70.90±4.89 0.30±0.03 9.17±0.93 14.54±1.42

10.67 10.47 10.18 8.73 11.67 6.89 9.27 10.12 9.76

83.87 91.17 93.74 88.91 99.05 110.78 75.43 91.67 90.89

Mean ± SD (ng/g) 2.64±0.33 80.58±4.74 122.32±7.73 1.35±0.10 28.28±2.94 61.08±4.31 0.42±0.03 9.57±0.76 16.34±1.42

CV (%) 12.49 5.88 6.32 7.57 10.38 7.05 6.5 7.94 8.66

Data are mean ± SD from quadruplicate samples at each spiked concentration of

imidacloprid.

31


Recovery (%) 82.63 100.72 95.56 84.58 70.71 95.44 104.44 95.66 102.14


Table 4. Eight tea samples analyzed by LFICS and UPLC-MS/MS (n=4) a Samples

S1 S2 S3 S4 S5 S6 S7 S8

Determined by LFICS (mg/kg) 0.030±0.002 0.080±0.01 ND b 0.125±0.01 ND ND ND ND

Determined by UPLC-MS/MS (mg/kg) 0.032±0.004 (imidacloprid) 0.082±0.01 (imidacloprid) ND 0.122±0.01 (imidacloprid) ND ND ND ND

a

All data are presented as mean±SD from quadruplicate well analysis of each sample.

b

ND means not determined.

Scheme 1. The direct competitive immunoassay of lateral flow immunochromatographic strip for the detection of neocotinoids in teas. Coating antigen was imidacloprid-OVA, analyte was imidacloprid, imidaclothiz or clothianidin, antibody was imidacloprid monoclonal antibody. 32


Page 32 of 34

Page 33 of 34


33



Graphical Abstract


Page 34 of 34

Quantum-Dot-Based Lateral Flow Immunoassay for Detection of

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