Subscriber access provided by FLORIDA STATE UNIV Article 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 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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Page 1 of 34 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 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Page 2 of 34 Page 3 of 34 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment mass liquid spectrometry Journal of Agricultural and Food Chemistry 59 Page 4 of 34 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 ACS Paragon Plus Environment 13 . A 14 . However, Page 5 of 34 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Page 6 of 34 103 (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 ACS Paragon Plus Environment (primary Page 7 of 34 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Page 8 of 34 Page 9 of 34 Journal of Agricultural and Food Chemistry 169 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 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Page 10 of 34 191 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 ACS Paragon Plus Environment Page 11 of 34 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Page 12 of 34 Page 13 of 34 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Page 15 of 34 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Page 16 of 34 Page 17 of 34 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Page 19 of 34 Journal of Agricultural and Food Chemistry 390 391 392 The authors have declared no conflict of interests. References (1) Zuo, Y.; Chen, H., Deng, Y. Simultaneous determination of catechins, 393 caffeine and gallic acids in green, oolong, black and pu-erh teas using 394 HPLC with a photodiode array detector. Talanta. 2002, 57, 307-16. 395 (2) Shao, X.; Liu, Z.; Xu, X.; Li, Z., Qian, X. 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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 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 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-line to C-line Ratio 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 ACS Paragon Plus Environment Page 27 of 34 Journal of Agricultural and Food Chemistry T/C T-line to C-line Ratio 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 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Green Tea 1.0 0.5 1.0 0.5 1E-3 0.01 0.1 1 10 Concentration of Imidacloprid (ng/mL) 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 T-line to C-line Ratio 1.5 Oolong Tea BB Buffer 10 - fold 20 - fold 40 - fold 80 - fold 160 - fold 1.5 T-line to C-line Ratio T-line to C-line Ratio 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 Concentration of Imidacloprid (ng/mL) 100 1E-3 0.01 Fig. 6. Matrix effects of different tea types on the assay performance. Bar, ±SD (n=4) 28 ACS Paragon Plus Environment 0.1 1 10 Concentration of Imidacloprid (ng/mL) 100 Page 29 of 34 Journal of Agricultural and Food Chemistry 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 <0.01 Thiamethoxam >1000 <0.01 Imidacloprid Structure Cl N CH2 N N NO 2 NH 29 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Page 30 of 34 Table 2. Comparison of the sensitivity of QD-based strip tests for the detection of imidacloprid, imidaclothiz, clothianidin with other reported immunoassays. Analysis method Analyte Sensitivity in assay Application buffer (ng/mL) sample Reference ELISA Clothianidin 25.6 (IC50) 10 Colloidal gold Clothianidin 8 (visual LOD) ELISA Imidaclothiz 58.16 (IC50) River water, soil, cabbage, tomato, greengrocery Tomato, pear, cabbage Chemiluminescenc e immunoassay ELISA Imidaclothiz 1.8 (IC10) 29 Clothianidin 4.4 (IC50) ELISA Imidacloprid Clothianidin 2.7 (IC50) >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 ACS Paragon Plus Environment 9 30 31 13 14 This article Page 31 of 34 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Recovery (%) 82.63 100.72 95.56 84.58 70.71 95.44 104.44 95.66 102.14 Journal of Agricultural and Food Chemistry 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 ACS Paragon Plus Environment Page 32 of 34 Page 33 of 34 Journal of Agricultural and Food Chemistry 33 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Graphical Abstract ACS Paragon Plus Environment Page 34 of 34
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