Portable Multiplex Immunochromatographic Assay for Quantitation of

Subscriber access provided by BUFFALO STATE

Food Safety and Toxicology

A portable multiplex immunochromatographic assay for the quantitation of two typical algae toxins based on dual-color fluorescence microspheres Huiyan Zhang, Jiaxun Luo, Natalia V. Beloglazova, Shupeng Yang, Ghulam Mujtaba Mari, Sarah De Saeger, Suxia Zhang, Jianzhong Shen, Zhanhui Wang, and Xuezhi Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00011 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

Journal of Agricultural and Food Chemistry

1

A Portable Multiplex Immunochromatographic Assay for

2

The Quantitation of Two Typical Algae Toxins Based on

3

Dual-color Fluorescence Microspheres

4

Huiyan Zhang,

5

Saeger, ‡ Ghulam Mujtaba Mari, † Suxia Zhang, † Jianzhong Shen, † Zhanhui Wang, †

6

and Xuezhi Yu*,†

7

†

8

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

9

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

†,‡

Jiaxun Luo,

†

Natalia Beloglazova,

‡

Shupeng Yang,

§

Sarah De

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

10

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

11

‡

12

Ottergemsesteenweg 460, 9000 Ghent, Belgium

13

§

14

Product Quality Supervision and Testing Center, Laboratory of Risk Assessment for

15

Quality and Safety of Bee Products, Ministry of Agriculture, Beijing 100093, People’s

16

Republic of China

Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University,

Institute of apicultural research, Chinese Academy of Agricultural Sciences, Bee

ACS Paragon Plus Environment


17

ABSTRACT: A multiplex immunochromatographic assay (ICA) based on dual-

18

color fluorescent microspheres (FMs) as sensitive label was developed for the first time.

19

Two typical algae toxins, microcystin-LR (MC-LR) and okadaic acid (OA), were

20

chosen as proof-of concept targets to evaluate the feasibility of this ICA format.

21

Commercial red and green colored FMs were selected to couple with monoclonal

22

antibodies as fluorescent probes. The use of dual-wavelength FMs as labels guaranteed

23

a lower consumption of material strips, lower sample volume, and shorter reaction time

24

without increasing the length of ICA strips. Under optimal conditions, the multiplex

25

FM-ICA could be completed in 20 min, and reached limits of detection (LOD) for the

26

simultaneous determination of MC-LR and OA in fish samples were 0.074 and 2.42

27

μg/kg, respectively. The developed technique was validated using artificially-spiked

28

and naturally-contaminated fish samples. Ultra-performance liquid chromatography-

29

tandem mass spectrometry (UHPLC-MS/MS) was used as confirmatory technique. In

30

summary, this portable ICAs detection mode based on dual-wavelength FMs provided

31

a reliable and sensitive on-site detection of multiple contaminants in food samples,

32

which opens a new field for application of FMs in food safety.

33

KEYWORDS: dual-color fluorescent microspheres, immunochromatographic assay,

34

multiplex detection, portable, algae toxins


Page 2 of 28

Page 3 of 28


INTRODUCTION

35



36

Nowadays, analytical chemistry has been facing new challenges and high requirements

37

in areas of medical diagnostics, environmental monitoring and food control 1. In these

38

fields, most analytes such as biomarkers, poisonous metal, mycotoxins, cyanotoxins,

39

antibiotics, pesticides and food additives could potentially co-exist in an individual

40

sample. This leads to an increasing need for cheap and fast solution for simultaneous

41

monitoring of two or more targets in one assay. Immunochromatographic assay (ICA),

42

also widely known as lateral flow device or strip test, as a combination of

43

chromatography and immunochemical reaction has been widely developed for point-

44

of-care testing due to its friendly user formats, low-costs and short assay times 2, 3. ICA

45

not only possesses the speed of homogeneous immunoassay but also keeps the

46

advantage of heterogeneous methods which can separate reacted and unreacted

47

compounds 4. In general, most developed ICAs could only detect a single target in one

48

assay which extremely limit the screening efficiency. Therefore, many different formats

49

of multiplex ICAs have been developed in past decades. However, part of them still

50

face some limitation to a certain degree1, 5.

51

One of the most common multiplex formats for rapid screening is

52

immunochromatographic assay where a test strip comprises several successive zones,

53

and each zone contains reagents specific for certain target

54

effective, fast, easy-to-perform and allows to work with small amounts of analyzed

55

samples, however interpretation of the results obtained by a multi-zone lateral flow

56

assay is not as convenient as that of a conventional single-parameter assay1. And the


5-10.

This approach is cost-


57

majority of multiplex ICA assays uses the same single-colored marker for each target

58

in different zones. This approach leads to interfering results between two adjacent zones.

59

Increasing a distance between these zones (and thereby increasing a length of the strip)

60

usually used to mitigate this issue; however, this strategy also means increased

61

consumption of analyzed samples and assay time. Actually, these drawbacks could be

62

overcame by using compatible but different colored markers in adjacent zones on a

63

single strip. For instance, Taranova et al. designed a “traffic light” ICA format with

64

three different water-soluble quantum dots whose emission maximum at either 525, 585,

65

or 625 nm, and this test system exhibited higher sensitivity for the detection of ofloxacin,

66

chloramphenicol, and streptomycin than ELISA using the same antibodies 1. Gharaat

67

et al. realized the simultaneous detection of two different toxins with different colors

68

of quantum dots 5. Besides quantum dots, multicolor latex beads is a new choice for

69

multiplex ICAs, Wang et al. choose crimson red, basic black and slate bule latex beads

70

developed a multiplex ICA for determination of three classes of antibiotic residues in

71

milk 11. Unlike common colloidal gold, gold nanoparticles whose color change from

72

red to purple-blue due to the strong surface plasmon resonance has been attract more

73

attention in the application of ICA, and they have been successfully applied in the

74

multiplex ICA strategy 12, 13. This approach based on multi-color probes facilitates the

75

identification of a corresponding analyte (each test zone has different color) and the

76

signal interference from adjacent lines could be avoided without extending the length

77

of strips.

78

Fluorescent microspheres (FMs), special polystyrene beads containing fluorescent


Page 4 of 28

Page 5 of 28


79

substance in their interior, have become a new perspective type of fluorescence labels.

80

The “multi-loaded” FMs not only solved the problem that single-dye molecules lack

81

enough chromosphere signal intensity, but also displayed many other advantages such

82

as stable configuration and high photostability

83

widely employed as labels in biomedical, disease diagnosis, food safety and genomics

84

16-18.

85

example, Li et al. 7 and Wang et al. 8 used red FMs realized the simultaneous detection

86

of three different macrolides and three different β-agonist residues in one strip,

87

respectively. However, it is a pity that only FMs with red emission light were used in

88

these multiple ICAs and no reports describing the use of different colored FMs in ICA

89

has been published so far.

14, 15.

In recent years, FMs has been

In addition, few multiplex ICAs based on FMs have been already reported, for

90

Microcystin-LR (MC-LR) and okadaic acid (OA) are typical algae toxins with

91

worldwide distribution, which are produced by cyanobacteria and dinoflagellates,

92

respectively. Although MC-LR and OA have different chemical structures, both could

93

cause tumor promotion and immunotoxicity by inhibiting the activity of protein

94

phosphatases 1 and 2A which are two key enzymes in cellular processes 19, 20. Human

95

beings are most likely to expose to MC-LR and OA by consuming of contaminated

96

drinking water and food. Thus, the simultaneous detection of these toxins in food and

97

drinks is very necessary. In present study, a unique multiplexed ICA based on dual-

98

wavelength FMs was developed and evaluated. Two typical algae toxins, MC-LR and

99

OA, were adopted as proof-of concept targets in the developed ICAs. To the best of our

100

knowledge, this is the first report describing a multiplex ICA based on dual-color FMs,



Page 6 of 28

101

and we hope this assay will open a new field in the application of fluorescence

102

microspheres for ICAs.

103



104

Reagents and apparatus. Carboxylate-modified microspheres, 2% solids, 200 nm, red

105

(RFM, 580/605, λex/λem), yellow-green (GFM, 505/515, λex/λem) and blue (BFM,

106

365/415, λex/λem) fluorescent microspheres were obtained from ThermoFisher

107

Scientific

108

hydrochloride (EDC), N-hydroxysuccinimide (NHS), phosphate buffer saline (PBS)

109

and 2-(N-morpholino) ethanesulfonic acid (MES) were supplied by Sigma-Aldrich (St.

110

Louis, MO, USA). Two monoclonal antibodies (mAbs) against MC-LR and OA were

111

recently produced by our group 21. MC-LR and OA (purity of ≥ 95% by HPLC) was

112

obtained from Taiwan Algal Science Inc. (Taiwan, China). The nitrocellulose filter

113

membrane (HF13520s25) were purchased from Millipore Corp. (Bedford, MA, USA).

114

Sample pad (CH37K) and absorbance pad (SB08) were provided by Shanghai Jinbiao

115

Biotechnology Co., Ltd. (Shanghai, China). Quantitative analysis of FM-ICA was

116

performed using ESE Quant reader, which were purchased from QIAGEN (Dusseldorf,

117

Germany).

118

Preparing of hapten-protein (antigen) conjugate. Both MC-LR-BSA and OA-BSA

119

conjugates were synthesized with a modification of the activated ester method 21, 22, and

120

the detailed description could be found in the supporting information.

121

Labeling of specific antibodies with FMs. The EDC-mediating activated ester method

MATERIALS AND METHODS

(MA,

USA);

1-ethyl-3-(3-(dimethylamino)


propyl)

carbodiimide

Page 7 of 28


122

was used to couple the carboxyl modified FMs with the mAb. The principle of the

123

reaction is presented on Figure 1(A). First, 15 μL of 2% FMs were suspended in 1 mL

124

MES buffer solution (0.05 M, pH=6), and EDC and NHS in MES solution were added,

125

and the mixture was stirred for 30 min. The mAb (0.2 mg/mL) was added to the

126

activated FMs, and the mixture was stirred for additional 30 min. Afterwards, 20 μL of

127

BSA in MES buffer (20%, w/v) were added into the mixture, and left for 15 min under

128

stirring. At last the mixture was centrifuged for 10 min (10000 rpm), and the

129

precipitates was washed with 1 mL PBS buffer for twice, then the precipitates was re-

130

suspended in 200 μL of PBS (0.01 M, pH 7.4) and ultrasonicated. All the conjugation

131

steps were occurred at room temperature.

132

Preparation of multi-ICA strips. The multi-component ICA strips were consisted with

133

an absorbent pad, a nitrocellulose membrane (NC membrane), a sample pad and a

134

backing pad as shown in Figure 1(B). The NC membrane was coated with two different

135

kinds of antigens and goat anti-mouse IgG antibody (second antibody), which formed

136

two test lines and one control line, with the dispense platform (ZX1000, US, Biodot) at

137

a jetting rate of 0.7 μL/cm. The membranes were dried at 37 °C for 2 h and stored in a

138

desiccator. Sample pad was impregnated with PBS (0.01 M, containing 0.05% sodium

139

azide and 0.05% Tween 20) and dried at 37 °C for 1 day. Then, absorbent pad,

140

nitrocellulose filter membrane and sample pad were pasted onto the packing pad

141

successively, and make sure both sample pad and absorbent pad overlapped a 2 mm

142

section of nitrocellulose filter membrane. Finally, the assembled backing was cut into

143

4 mm wide strips and kept in desiccator at room temperature (RT).



144

Qualitative and quantitative multi-ICAs procedure. A mixture of two multi-color FM-

145

mAb conjugates (each of them were 2 μL) and 120 μL of a sample solution (or standard

146

solutions) were mixed into a 96-well microtiter plate and incubated for 3 min at RT.

147

Then, the strip was vertically inserted into the well to absorb the mixture, and it was

148

removed after capillary migration process about 15 min at 37 °C in an incubator. The

149

results of ICAs could be either qualitatively judged with the naked eye under ultraviolet

150

(UV) light or quantitatively detected by measuring the fluorescence signal intensities

151

using portable lateral flow reader with excitation/emission wavelengths set at 580 nm

152

/605 nm for the red FMs and 470/520 nm for yellow-green FMs, respectively.

153

Fish samples preparation for multiplex ICA and UHPLC-MS/MS methods. Samples

154

preparation for multiplex ICA strips: 5 mL methanol was added to five gram

155

homogenized fish meat samples, and the mixture was shaken for 1 min on a vortex

156

shaker and with ultrasonic treatment for 10 min. Then, it was centrifuged at 10000 rpm

157

for 10 min, supernatant was reserved and this process was repeated twice. Then, two

158

supernatants were evaporated to dryness with a stream of nitrogen at 40 °C. The residue

159

was dissolved in 5 mL of PBS and used for detection.

160

Samples preparation for UHPLC-MS/MS method: One gram homogenized fish meat

161

samples were extracted by methanol (5 mL). After centrifugation, the collected

162

supernatant was evaporated, and then it was re-dissolved with 3 mL of 5 % acetonitrile

163

(acetonitrile: water, 5:95, v/v). Afterward, the cleaned-up by solid phase extraction

164

(SPE) on an Oasis HLB cartridge (3 mL, 60 mg) was adopted. Firstly, 3 mL of methanol

165

and 3 mL of water ere load on the SPE column, sequentially. Then the cartridge was


Page 8 of 28

Page 9 of 28


166

rinsed by 3 mL of 5 % methanol (methanol: water, 5:95, v/v) and followed by elution

167

using 3 mL of methanol. The elution was evaporated to dryness under nitrogen and re-

168

dissolved with 1 mL of 15 % acetonitrile (acetonitrile: water, 15:85, v/v), which was

169

then analyzed by an in-house validated UHPLC-MS/MS method. Detail information

170

about the proposed UHPLC-MS/MS method was indicated in supporting information,

171

such as LC and MS conditions, MRM parameters (Table S1), and chromatograms of

172

quantification ions for MC-LR and OA (Figure S1).

173

Validation of developed ICA method. The MC-LR and OA-blank fish samples

174

confirmed by UHPLC-MS/MS were obtained from the National Reference Laboratory

175

for Veterinary Drug Residues (Beijing, China). And it was spiked with different

176

volumes MC-LR and OA mixed standards standing for 15 min. For the recoveries tests,

177

MC-LR at the final concentration level of 0.5, 2, and 5 μg/Kg were spiked, and OA at

178

the final concentration level of 5, 10, and 20 μg/Kg were spiked. Then, the recoveries

179

were calculated comparing the spiked and found MC-LR and OA in the samples,

180

respectively. The validity of ICA method was verified by analyzing six different fish

181

samples collected from MerryMart, Haidian District (Beijing, China), and these fish

182

samples were spiked with MC-LR and OA mixed standards at the level of 2.5 and 30

183

μg/Kg, respectively. The detection results of six fish samples were further compared

184

and validated with the analysis results acquired with UHPLC-MS/MS method. All the

185

fish samples were treated with methods mentioned above.

186



RESULT AND DISCUSSION



187

Principle of the multiplex FM-ICA. The multiple FM-ICA was developed according to

188

the competitive inhibitory interaction between the free analysis targets in sample and

189

the hapten–protein conjugates which were spatially coated on the NC membrane 9. As

190

showed in Figure 1(C), two FMs with different emission peaks were used to label the

191

antibodies that allowed to clearly distinguish each line on the test strip. If no MC-LR

192

and OA were present in samples, the FMs-mAb conjugates bound to the antigens

193

adsorbed on the test lines and formed the colored bands. If the analytes presented in

194

samples, they bound to the FMs-mAb conjugates firstly and form FMs-mAb-analyte

195

complexes in the well. Then, the complexs migrated on the strips by capillary action

196

and diminished the color intensity of test lines. According to the principle described

197

above, the fluorescence intensity of the test lines was reversely proportional to

198

concentration of the targets in analyzed samples, and gradually decreased to zero as the

199

concentration of analyte increased. The concentration which resulted no color with

200

naked eyes on the test lines was defined as cut-off values, and it was used to semi-

201

quantitative detection for on-site test. Regardless of presence of targets or not in

202

samples, FM labelled mAb conjugates would pass through test lines and react with the

203

second antibody on control line which was used to ensure the validity of the detection.

204

Preparation of the FM-mAb conjugates. In the present study, three FMs (red, blue,

205

yellow-green) with different excitation and emission wavelengths were evaluated at

206

first in order to select optimum two FMs as probes. We checked the excitation and

207

emission spectra of these three FMs on the internet, and found that there are overlap

208

between the excitation spectra of GFM and the emission spectra of RFM, however,


Page 10 of 28

Page 11 of 28


209

considering these two colored-FMs were separated at different positions on the strips,

210

this interference could be negligible (in Figure 2 (A)). Subsequently, MC-LR mAbs

211

was chosen as the target to conjugate with three FMs and to develop ICAs, respectively.

212

On surface of NC membrane, MC-LR (10 ng/mL) and MC-LR-BSA coated on the test

213

lines compete to bind with different colors of FMs labelled antibody. As shown in

214

Figure 2 (B), in comparison with BFM, both RFM and GFM displayed brighter

215

fluorescence and more obvious contrast with the blue background under UV light. After

216

adding 10 ng/mL MC-LR in buffers, RFM labeled antibody displayed highest

217

sensitivity with lowest fluorescence intensities on test line, and there are no obvious

218

difference between GFM and BFM. Thus, RFM and GFM were chosen as the multi-

219

labels in ICA for the next steps, which allowed users to clearly distinguish between

220

each line on the strips.

221

Activated ester technique was selected to conjugate antibodies and FMs, in which

222

EDC and NHS were used to active the carboxyl groups on the surface FMs. Choosing

223

the optimal concentrations of EDC/NHS is very important for this reaction, as low

224

coupling efficiency could be caused by insufficiency of EDC and NHS, and severe

225

aggregation of conjugates which is not suitable to move on the strips could appear with

226

excess EDC and NHS 23. In this assay, the amount of FMs were fixed with 15 μL during

227

the optimization procedures due to the lacking of clear concentration of commerical

228

FMs. We found that the optimum amount of EDC/NHS was 1 μg where they could

229

obtained the brightest fluorescence on the strips (Figure S2 in the supporting

230

information). In order to achieve the best sensitivity and the required visibility, the



231

optimal volumes of MC-LR and OA antibodies (whose original concentration were

232

23.99 mg/mL and 20.04 mg/mL, respectively) for conjugation both were 50 μL with

233

the highest peak area and inhibition ration in Figure 3 (A) and (B). In addition, pH value

234

of the reaction system has a great influence on the sensitivity, stability and fluorescent

235

intensity of ICAs7, 15. The flocculent precipitate appeared in the tube in an acidic or

236

alkaline environment when pH≥8 or ≤4 during the FMs and antibody conjugation

237

procedure. When the conjugates obtained under different pH conditions were used to

238

build strips (Figure S3 in the supporting information), the fluorescence intensity of

239

control line was significantly enhanced with pH increased from 4 to 8, and no

240

fluorescence were shown on the test strips when pH=4. ICA showed highest sensitivity

241

and high fluorescence intensity when pH was 6, therefore, the pH of the reaction system

242

was chosen to be 6. This results is consistent with previous reports that the optimum

243

pH for EDC reaction was 4–6, and NHS was used to stabilize the reaction environment

244

and improve coupling efficiency 24, 25.

245

Optimization of FM-ICA. In this multiplex FM-based ICA format, four different

246

position combinations could be formed as shown in Figure 4(A) as two different colors

247

of FMs and two kinds of coating antigen are used. Optimal composition on the strips

248

should provide high sensitivity and bright fluorescence signal. As presented in Figure

249

4(B), we found that the fluorescence intensity of test lines was much brighter if MC-

250

LR antigen was coated on T-1 line near the bottom of the strip. Besides, both of the

251

probes displayed brighter fluorescence when RFM was used to label MC-LR antibody

252

and GFM was used to label OA antibody. Except the amount of antibody, hapten-


Page 12 of 28

Page 13 of 28


253

protein conjugates coated on the strip also has a great influence on the analytical

254

performance. According to the peak area and inhibition ration which were displayed on

255

Figure 3 (C) and (D), the optimal concentration of MC-LR–BSA and OA-BSA coated

256

on the strips were 0.56 mg/mL and 1.16 mg/mL, respectively.

257

Performance of FM-ICA. Fluorescent intensity of test lines on ICAs decreased with

258

the increased concentration of two analytes, qualitative and quantitative results can be

259

obtained from this FMs-based ICAs. Under the optimal conditions, different

260

concentrations of MC-LR and OA in blank fish tissues were applied, absence of the

261

targets in the fish tissues was confirmed by an ultra-performance liquid

262

chromatography-tandem mass spectrometry method (UHPLC-MS/MS) which were

263

developed according to previous reports 26-28 and detailed information could be found

264

in the supporting information. As shown in Figure 5(A), in the absence of MC-LR and

265

OA in the samples, two test lines of red and green, one control line of yellow were

266

observed by the naked eye under an UV light. With increasing concentrations of each

267

analyte, the fluorescence of the corresponding test line gradually decrease. The

268

fluorescence completely disappeared when the MC-LR and OA concentration were

269

2.22 μg/kg and 33.33 μg/kg, respectively. Therefore, 2.2 μg/kg and 33.3 μg/kg were

270

defined as the cutoff values for MC-LR and OA in this multiplex FM-ICA, respectively.

271

In this dual-wavelength FMs based ICA, the contrasting marker colors enable the user

272

to easily interpret the test results corresponding to each particular analyte.

273

The quantitative detection of the fluorescence intensities on the ICAs strips was

274

performed by a portable fluorescence reader, and the peak area of the test lines which



275

were detected by this fluorescence reader were used to quantify the concentration of

276

MC-LR or OA in fish samples. The calibration curves for detection of the analytes were

277

set up in Figure 5(B), and the analytical performances were listed in Table S2. The IC50

278

values were of 0.68 and 11.07 μg/kg for MC-LR and OA, respectively. The LODs for

279

MC-LR and OA fish samples was experimentally defined as the concentration that

280

corresponds to 10% inhibition of the peak area signal (IC10) 29, which were calculated

281

as 0.074 and 2.42 μg/kg for MC-LR and OA, respectively.

282

Recoveries and reproducibility of FM-ICA. This developed FM-ICA were validated

283

using artificially-spiked blank fish samples tests as shown in Table 1. The recoveries

284

of blank fish samples were estimated by spiking with low, moderate and high

285

concentration of MC-LR and OA standards before treating with methods described

286

above. The average recovery values (n=3) of MC-LR and OA in the FM-based ICA

287

ranged from 89.4–95.2% and 94.8–109.4%, respectively. The ICA also exhibited the

288

high reproducibility, for the relative standard deviations for toxins detection in the

289

working range of the calibration curves were 3.9–9.1% for MC-LR and 4.3–10.4% for

290

OA. In order to further evaluate the developed ICA, six blind fish samples were

291

analyzed in triplicate and validated with the UHPLC-MS/MS method, as it could be

292

found in a Table 2, there is no obvious difference in the results between the ICA and

293

UHPLC–MS/MS. The two methods displayed a high consistency with the coefficient

294

(R2) of 0.99 for the spiked fish samples. These result confirmed that the developed FM-

295

ICA could be used for analysis of MC-LR and OA in fish samples with the good

296

accuracy and precision.


Page 14 of 28

Page 15 of 28


297

In summary, a simple, portable and sensitive multiplex ICA was designed for the

298

simultaneous detection of two different targets using the dual-wavelength FMs as labels.

299

The multi-colored FMs allows users easy to qualitatively and quantitatively identify

300

target analytes based on the color of the lines. Besides, the use of different wavelength

301

of FMs in one strip was proved feasible, and the effect of fluorescence interference

302

from adjacent lines could be avoided. Two typical algae toxins, MC-LR and OA, were

303

employed to illustrate this concept. The developed ICAs based on dual-wavelength

304

FMs displayed high sensitivity, the limits of detection of MC-LR and OA were 0.074

305

and 2.42 μg/kg, respectively. The ICA could be used for detection of MC-LR and OA

306

in fish samples with the high recoveries of more than 89%. As a confirmation technique,

307

the UHPLC-MS/MS was used and no big differences were found in six blind fish

308

samples. The developed ICA represents a new strategy for the multiplex detection based

309

on FMs and displayed a high potential use of FMs in immunoassays.

310



311

ICA, immunochromatographic assay; FMs, fluorescent microspheres; MC-LR,

312

microcystin-LR; OA, okadaic acid; LOD, limits of detection; UHPLC-MS/MS, ultra-

313

performance liquid chromatography-tandem mass spectrometry; RFM, red fluorescent

314

microspheres; GFM, yellow-green fluorescent microspheres; BFM, blue fluorescent

315

microspheres; EDC, 1-ethyl-3-(3-(dimethylamino) propyl) carbodiimide hydrochloride;

316

NHS, N-hydroxysuccinimide; PBS, phosphate buffer saline (PBS); MES, 2-(N-

317

morpholino) ethanesulfonic acid; mAbs, monoclonal antibodies; UV, ultraviolet.

ABBREVIATIONS USED



ACKNOWLEDGMENTS

318



319

This project was supported by Beijing Keypoint Research and Invention Program

320

(D171100008317003) and National Key R&D Program of China (2018YFC1602600).

321



322

Supporting Information

323

The Supporting Information is available free of charge on the ACS Publications website.

324

Additional information including the steps of how to prepare of hapten-protein

325

conjugate; The characteristic chromatograms of quantification ions for MC-LR and OA

326

using UHPLC-MS/MS (Figure S1); optimization of the amount of EDC/NHS (Figure

327

S2); conjugation of FMs and mAb with different pH values (Figure S3); Summary of

328

MRM parameters for the detection of MC-LR and OA using UHPLC-MS/MS (Table

329

S1); Analytical characteristics parameters of standard curves of FM-ICA (Table S2).

330



331

Corresponding Authors

332

*Phone: +86-10-6273 4565, E-mail: [email protected] (Z.Y.)

333

Notes

334

The authors declare no competing financial interest.

ASSOCIATED CONTENT

AUTHOR INFORMATION

335


Page 16 of 28

Page 17 of 28


336



337

1.

REFERENCES Taranova, N. A.; Berlina, A. N.; Zherdev, A. V.; Dzantiev, B. B., 'Traffic light'

338

immunochromatographic test based on multicolor quantum dots for the simultaneous detection of

339

several antibiotics in milk. Biosens. Bioelectron. 2015, 63, 255-261.

340

2.

341 342

Bahadir, E. B.; Sezginturk, M. K., Lateral flow assays: Principles, designs and labels. Trac-Trend Anal. Chem. 2016, 82, 286-306.

3.

Sheng, W.; Chang, Q.; Shi, Y. J.; Duan, W. X.; Zhang, Y.; Wang, S., Visual and fluorometric lateral

343

flow immunoassay combined with a dual-functional test mode for rapid determination of

344

tetracycline antibiotics. Microchim. Acta. 2018, 185.

345

4.

346 347

Dzantiev, B. B.; Byzova, N. A.; Urusov, A. E.; Zherdev, A. V., Immunochromatographic methods in food analysis. Trac-Trend Anal. Chem. 2014, 55, 81-93.

5.

Gharaat, M.; Sajedi, R. H.; Shanehsaz, M.; Jalilian, N.; Mirshahi, M.; Gholamzad, M., A dextran

348

mediated multicolor immunochromatographic rapid test strip for visual and instrumental

349

simultaneous detection of Vibrio cholera O1 (Ogawa) and Clostridium botulinum toxin A.

350

Microchim Acta. 2017, 184, 4817-4825.

351

6.

Han, S.; Zhou, T.; Yin, B.; He, P., A sensitive and semi-quantitative method for determination of

352

multi-drug residues in animal body fluids using multiplex dipstick immunoassay. Anal. Chim. Acta.

353

2016, 927, 64-71.

354

7.

Li, X. M.; Shen, J. Z.; Wang, Q.; Gao, S. X.; Pei, X. Y.; Jiang, H. Y.; Wen, K., Multi-residue

355

fluorescent microspheres immunochromatographic assay for simultaneous determination of

356

macrolides in raw milk. Anal. Bioanal. Chem. 2015, 407, 9125-9133.

357

8.

358 359

Wang, P. L.; Wang, Z.; Su, X. O., A sensitive and quantitative fluorescent multi-component immuno-chromatographic sensor for beta-agonist residues. Biosens. Bioelectron. 2015, 64, 511-516.

9.

Xing, C. R.; Liu, L. Q.; Song, S. S.; Feng, M.; Kuang, H.; Xu, C. L., Ultrasensitive

360

immunochromatographic assay for the simultaneous detection of five chemicals in drinking water.

361

Biosens. Bioelectron. 2015, 66, 445-453.

362 363 364

10. Zhang, D. H.; Li, P. W.; Zhang, Q.; Li, R.; Zhang, W.; Ding, X. X.; Li, C. M., A naked-eye based strategy for semiquantitative immunochromatographic assay. Anal. Chim. Acta. 2012, 740, 74-79. 11. Wang, C.; Li, X. M.; Peng, T.; Wang, Z. H.; Wen, K.; Jiang, H. Y., Latex bead and colloidal gold



365

applied in a multiplex immunochromatographic assay for high-throughput detection of three classes

366

of antibiotic residues in milk. Food Control 2017, 77, 1-7.

367

12. Wang, Q.; Liu, Y.C.; Wang, M.Y.; Chen, Y.J.; Jiang, W.; A multiplex immunochromatographic test

368

using gold nanoparticles for the rapid and simultaneous detection of four nitrofuran metabolites in

369

fish samples. Anal. Bioanal. Chem. 2018, 410, 223–233.

370

13. Di Nardo, F.; Baggiani, C.; Giovannoli, C.; Spano, G.; Anfossi, L., Multicolor

371

immunochromatographic strip test based on gold nanoparticles for the determination of aflatoxin

372

B1 and fumonisins. Microchim. Acta. 2017, 184, 1295-1304.

373

14. Langenhorst, R. J.; Lawson, S.; Kittawornrat, A.; Zimmerman, J. J.; Sun, Z.; Li, Y. H.; Christopher-

374

Hennings, J.; Nelson, E. A.; Fang, Y., Development of a fluorescent microsphere immunoassay for

375

detection of antibodies against porcine reproductive and respiratory syndrome virus using oral fluid

376

samples as an alternative to serum-based assays. Clin. Vaccine. Immunol. 2012, 19, 180-189.

377

15. Wang, Z.; Li, H.; Li, C.; Yu, Q.; Shen, J.; De Saeger, S., Development and application of a

378

quantitative fluorescence-based immunochromatographic assay for fumonisin b1 in maize. J. Agric.

379

Food Chem. 2014, 62, 6294-8.

380

16. Pan, F. G.; Fang, Z.; Meng, R. Z.; Qin, Y. N.; Ju, W.; Liu, J. H.; Wu, H., Improving the sensitivity

381

of fluorescent microsphere immunoassays for the simultaneous detection of foodborne pathogens

382

using dimethylacetamide. Anal. Lett. 2013, 46, 2203-2212.

383

17. Wang, Y.; Ning, B. A.; Peng, Y.; Bai, J. L.; Liu, M.; Fan, X. J.; Sun, Z. Y.; Lv, Z. Q.; Zhou, C. H.;

384

Gao, Z. X., Application of suspension array for simultaneous detection of four different mycotoxins

385

in corn and peanut. Biosens. Bioelectron. 2013, 41, 391-396.

386

18. Zhou, J.; Zhu, K.; Xu, F.; Wang, W.; Jiang, H.; Wang, Z.; Ding, S., Development of a microsphere-

387

based fluorescence immunochromatographic assay for monitoring lincomycin in milk, honey, beef,

388

and swine urine. J. Agric. Food Chem. 2014, 62, 12061-6.

389

19. Nishiwaki-Matsushima, R.; Ohta, T.; Nishiwaki, S.; Suganuma, M.; Kohyama, K.; Ishikawa, T.;

390

Carmichael, W. W.; Fujiki, H., Liver tumor promotion by the cyanobacterial cyclic peptide toxin

391

microcystin-LR. J. Cancer Res. Clin. Oncol. 1992, 118, 420-4.

392

20. Suganuma, M.; Fujiki, H.; Suguri, H.; Yoshizawa, S.; Hirota, M.; Nakayasu, M.; Ojika, M.;

393

Wakamatsu, K.; Yamada, K.; Sugimura, T., Okadaic acid: an additional non-phorbol-12-

394

tetradecanoate-13-acetate-type tumor promoter. Proc. Natl. Acad. Sci. USA 1988, 85, 1768-71.


Page 18 of 28

Page 19 of 28


395

21. Yang, H. J.; Dai, R.; Zhang, H. Y.; Li, C. L.; Zhang, X. Y.; Shen, J. Z.; Wen, K.; Wang, Z. H.,

396

Production of monoclonal antibodies with broad specificity and development of an immunoassay

397

for microcystins and nodularin in water. Anal. Bioanal. Chem. 2016, 408, 6037-6044.

398

22. Matsuura, S.; Kita, H.; Takagaki, Y., Specificity of mouse monoclonal anti-okadaic acid antibodies

399

to okadaic acid and its analogs among diarrhetic shellfish toxins. Biosci. Biotechnol. Biochem. 1994,

400

58, 1471-5.

401

23. Foubert, A.; Beloglazova, N. V.; De Saeger, S., Comparative study of colloidal gold and quantum

402

dots as labels for multiplex screening tests for multi-mycotoxin detection. Anal. Chim. Acta. 2017,

403

955, 48-57.

404

24. Lee, J. M.; Edwards, H. H. L.; Pereira, C. A.; Samii, S. I., Crosslinking of tissue-derived

405

biomaterials in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). J. Mater. Sci-Mater. M.

406

1996, 7, 531-541.

407

25. Damink, L. H. H. O.; Dijkstra, P. J.; vanLuyn, M. J. A.; vanWachem, P. B.; Nieuwenhuis, P.; Feijen,

408

J., In vitro degradation of dermal sheep collagen cross-linked using a water-soluble carbodiimide.

409

Biomaterials 1996, 17, 679-684.

410

26. Shen, Q.; Gong, L.K.; Baibado, J.T.; Dong, W.; Wang, Y.X.; Dai, Z.Y.; Cheung, H.Y.; Graphene

411

based pipette tip solid phase extraction of marine toxins in shellfish muscle followed by UHPLC-

412

MS/MS analysis. Talanta 2013 116,770-775.

413

27. Suzuki, T.; Quilliam, M. A., LC-MS/MS analysis of diarrhetic shellfish poisoning (DSP) toxins,

414

okadaic acid and dinophysistoxin analogues, and other lipophilic toxins. Anal. Sci. 2011, 27, 571-

415

84.

416

28. Zhang, H. Y.; Yang, S. P.; Beier, R. C.; Beloglazova, N. V.; Lei, H. T.; Sun, X. L.; Ke, Y. B.; Zhang,

417

S. X.; Wang, Z. H., Simple, high efficiency detection of microcystins and nodularin-R in water by

418

fluorescence polarization immunoassay. Anal. Chim. Acta. 2017, 992, 119-127.

419

29. Murtazina, N.R.; Eremin, S.A.; Mozoleva, O.V.; Everest, S.J.; Brown, A.J.; Jackman, R.; Fluorescent

420

polarization immunoassay for sulphadiazine using a high specificity antibody. Int. J. Food Sci. Tech.

421

2004, 39, 879-889



Page 20 of 28

Figure captions Figure 1. (A) Schematic synthesis of the FMs-labelled conjugates; (B) diagram of multiplex ICA strip and coating position for MC-LR-BSA and OA-BSA; (C) Schematic representation of the competitive FMs-based ICA for the simultaneousl detection of MC-LR and OA. Figure 2. (A) Excitation and emission spectra of the red, yellow-green and blue FMs from

the

fluorescence

spectra

viewer,

which

is

available

on:

https://www.thermofisher.com/be/en/home/life-science/cell-analysis/labelingchemistry/fluorescencespectraviewer.html. (B) Selection of three different FMs for multiplex ICAs with MC-LR corresponding reagents. Figure 3. The optimization of the amount of immunoreagents of the ICA, (A) the volume of MC-LR mAb; (B) the volume of OA mAb; (C) the concentration of MCLR-BSA; (D) the concentration of OA-BSA. Figure 4. (A) The diagram of different position combinations consist with two different FMs and two different targets, (B) the picture of four different combinations, the left one is blank buffer, the right one is mixture of MC-LR and OA standard (100 ng/mL) Figure 5. (A) Fluorescent image of the FM-ICA acquired under UV light. (B) Calibration curves for the quantitative detection by a portable fluorescence FM-ICA reader.


Page 21 of 28


Table 1. Summary of the validated parameters of recovery and coefficient of variation (CV) for MC-LR and OA by spiking low, medium and high concentration level in blank fish samples (n = 3) using the developed FM-ICA. Analyte

Original level (μg/kg)

Spiked level (μg/kg)

Detected (μg/kg)

Recovery (%)

CV (%)

MC-LR

Not found

0.5

0.476

95.2

3.9

Not found

1

0.894

89.4

9.1

Not found

2

1.86

93.1

5.9

Not found

5

5.27

105.3

10.4

Not found

10

10.9

109.4

7.9

Not found

20

18.9

94.7

4.3

OA



Page 22 of 28

Table 2. Comparison of analytical results for MC-LR and OA using FM-ICA and LC-MS/MS in six blind fish samples (n=3) Sample No.

1 2 3 4 5 6

FM-ICA (qualitative) MC-LR No Spiked with spiked 2.5 (μg/kg) + + + ± + + -

No spiked

-

OA Spiked with 30 (μg/kg) + + + + + +

FM-ICA (quantitative) No spiked ND ND ND ND ND ND

MC-LR Spiked with 2.5 (μg/kg) 2.24±0.21 2.19±0.15 2.13±0.25 1.99±0.23 2.59±0.16 2.07±0.25

OA No Spiked with spiked 30 (μg/kg) ND 28.1±1.45 ND 26.5±1.06 ND 29.6±2.37 ND 24.5±0.85 ND 27.9±1.76 ND 23.4±0.96

LC-MS/MS MC-LR No Spiked with spiked 2.5 (μg/kg) ND 2.43±0.16 ND 2.61±0.30 ND 2.35±0.14 ND 2.45±0.22 ND 2.67±0.13 ND 2.38±0.20

Note: - : negative results, absence of toxins; +: positive results, exist of toxins; ±: weakly positive, light fluorescence intensity is observed; ND: not detected


No spiked ND ND ND ND ND ND

OA Spiked with 30 (μg/kg) 31.2±2.04 28.8±1.26 29.4±2.16 30.7±1.58 28.9±1.92 29.4±1.63

Page 23 of 28


Figure 1



Figure 2


Page 24 of 28

Page 25 of 28


Figure 3



Figure 4


Page 26 of 28

Page 27 of 28


Figure 5



Graphical Abstract


Page 28 of 28

Portable Multiplex Immunochromatographic Assay for Quantitation of

Recommend Documents