A Dual-Color Quantum Dots Encoded Frit-Based Immunoassay for

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A Dual-Color Quantum Dots Encoded Frit-based Immunoassay for Visual Detection of Aflatoxin M1 and Pirlimycin Residues in Milk Wenxiao Jiang, Natalia V. Beloglazova, Pengjie Luo, Ping Guo, Guimiao Lin, and Xiaomei Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05337 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 13, 2017

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Journal of Agricultural and Food Chemistry

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A Dual-Color Quantum Dots Encoded Frit-based Immunoassay for

2

Visual Detection of Aflatoxin M1 and Pirlimycin Residues in Milk

3 4

Wenxiao Jiang†, Natalia V. Beloglazova‡, Pengjie Luo§, Ping Guo#, Guimiao Lin†,

5

and Xiaomei Wang*,†

6 7

†

8

Health Sciences Center, Shenzhen 518060, China

9

‡

Department of Physiology, School of Basic Medical Sciences, Shenzhen University

Laboratory of Food Analysis, Department of Bioanalysis, Ghent University,

10

Harelbekestraat 72, B-9000, Ghent, Belgium

11

§

12

National Center for Food Safety Risk Assessment, Beijing 100021, China

13

#

14

Nanchang 330038, China

Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China

Technology Center of JiangXi Entry-Exit Inspection and Quarantine Bureau,

15 16 17 18 19 20

∗ Corresponding author. Tel: +86-755-8667-1936; Fax: +86-755-8667-1906; E-mail:

21

[email protected] (X. Wang).

1

ACS Paragon Plus Environment


22

Abstract

23

Mycotoxins and antibacterial agents are the main chemical hazards that lead to several

24

health problems. Nowadays, multiplex immunoassay is a primary goal throughout the

25

world. Here, aflatoxin M1 and pirlimycin were selected as models, and a novel dual

26

colorimetric encoded frit-based immunoassay was developed for simultaneously

27

screening of aflatoxin M1 and pirlimycin residues in milk. This multiplex frit-based

28

immunoassay combined two monoclonal antibodies in order to extend the spectrum of

29

analytes, and to enable detection of two classes of analytes in a single test. The cut-off

30

values were 0.02 µg/kg for aflatoxin M1 and 0.5 µg/kg for pirlimycin, which satisfied

31

the requirement to measure the maximum residue levels. The novel colorimetric

32

frit-based immunoassay has the advantage of high throughput, short analysis time,

33

reduced overall cost per assay, and can be used as a rapid screening technique for

34

simultaneously detecting aflatoxin M1 and pirlimycin residues in milk.

35

Key Words: aflatoxin M1; pirlimycin; immunoassay; milk; food safety

36

2


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Introduction

38

Nowadays, food safety problem due to contamination with mycotoxins and

39

antibacterial agents has been a growing concern among the government and

40

public.1,

41

produced by Aspergillus flavus and Aspergillus parasiticus.3, 4 Aflatoxin M1

42

(AFM1), the hydroxylated derivative of aflatoxin B1, is often found in milk

43

from animals fed with AFB1-contaminated feeds.5 Besides, the intensive

44

cultivation pattern for producing food animals routinely uses mass veterinary

45

drugs (i.e. pirlimycin, PIR) to prevent animal diseases and to improve growth

46

performance.6 Residues of these chemicals may enter the food chain by

47

non-compliance of withdrawal times. (Figure S1) The presence of these

48

chemical residues in milk constitutes a potential health hazard for humans due

49

to toxic reactions.7 In order to protect consumers from exposure to these

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chemical residues, many countries have established strict regulations, including

51

withdrawal times and maximum residue limits (MRLs) for AFM13 and PIR8.

2

Aflatoxins are highly toxic mycotoxins, secondary metabolites

52

Concern about food safety requires the development of rapid and sensitive

53

on-site tests, suitable for non-laboratory application.9 For screening purposes,

54

the immunoassay is advantageous compared to instrumental methods because

55

of its high throughput and rapid turnaround time.10 Multiplex immunoassays

56

allow analysis of several analytes with a single sample pretreatment avoiding

57

the use of several chemistries and methodologies for each analyte, saving time

58

and resources.11, 12 Some multiplex immunoassays were designed by placing 3



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immunoreagents, specific towards different analytes, on separate spots within

60

one test system.13, 14 Besides, different color emitting materials were also used

61

as fluorescent labels in the multiplex immunoassays.15 With new progress in

62

nanoscience and material science, more and more label techniques have been

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introduced for antibody-based immunoassays, such as lanthanide16, fluorescent

64

microspheres and quantum dots (QDs)17. QDs are characterized by a narrow,

65

size-tunable, symmetric emission spectrum, and quite high photochemical

66

stability, with multi-colored signal under ultraviolet light.18 QD-based

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immunoassays have shown many advantages such as high sensitivity caused by

68

their high photoluminescence brightness, rapid and easy detection of analytical

69

signal, and low sensitivity to environmental conditions.19

70

In this study, the immobilization of two different types of antibodies onto a

71

polyethylene frit for simultaneous fluorescent detection of AFM1 and PIR residues

72

was described. Green- and red-emitting QDs were encapsulated into liposomes and

73

used for synthesis of the labeled conjugates. The multiplex frit-based immunoassay

74

demonstrated good reliability for screening AFM1 and PIR residues in milk. Further,

75

the

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naturally-contaminated milk samples by liquid chromatography tandem mass

77

spectrometry (LC-MS/MS) methods. Compared with traditional immunoassays, the

78

frit-based immunoassay requires the least sample pretreatment, without the need for

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expensive equipment, and the results can be obtained within 30 minutes.

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Materials and Methods

developed

technique

was

validated

in

4


artificially-spiked

and

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Chemicals and apparatus

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AFM1 and other aflatoxins were supplied by Fermentek (Jerusalem, Israel).

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PIR and other lincosamide antibiotics were purchased from Toronto Research

84

Chemicals Inc. (Toronto, Canada). Bovine serum albumin (BSA), ovalbumin

85

(OVA), N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-3-ethyl

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carbodiimide (EDC) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

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Goat-anti-mouse

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Laboratories Inc. (West Grove, PA, USA). Polystyrene microplates were

89

purchased from Costar (Milpitas, CA, USA). Plastic tubes (Bond Elut reservoir,

90

1 mL) and polyethylene frits (1/4 in diameter) were supplied by Agilent

91

Technologies (Santa Clara, CA, USA).

92

Preparation of an antigen and development of ELISA

IgG

was

obtained

from

Jackson

Immuno

Research

93

AFM1 was first converted to AFM1-o-carboxymethyl-oxime (AFM1-oxime) by

94

the introduction of a reactive carboxyl group from carboxymethoxylamine

95

hemihydrochloride. The synthesized hapten was bound to BSA using the

96

N-hydroxysuccinimide ester method.3 The PIR hapten was covalently attached to

97

BSA by use of a sodium periodate (NaIO4) reduction method.8 The procedures

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employed for generation of the MAbs and the development of ELISAs for AFM1 and

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PIR were similar to our previous work.20

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Preparation of the QDs-labelled conjugates

101

The synthesis of red- and green-emitting CdSe/ZnS core/shell QDs were

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performed according to previously published research.21 The liposomes loaded with 5



103

QDs (LQDs) were prepared by a thin-film hydration method based on previous

104

reports.22 Briefly, lipoid S75 (94 µmol) and QD (1 nmol) were dissolved in 1 mL of

105

chloroform. After removing the organic solvent by rotary evaporation at 45 °C, 6 mL

106

of water were added to the dry lipid film. Then, the mixture was vigorously stirred in

107

a water bath at 45°C for 30 min and sonicated for 5 min. The LQDs were separated

108

and re-dissolved in PBS after centrifugation at 20 000 × g for 10 min at room

109

temperature. The conjugation of the coating antigen with LQDs via N-succinimidyl

110

3-(2-pyridyldithio) propionate was performed according to methods described in our

111

previously research.13(Figure S2) These conjugates were stored at 4 °C before use.

112

Preparation of the functionalized polyethylene frit

113

First, the surface of the polyethylene frit was covered with a glutaraldehyde (GA)

114

monolayer according to previously published methods23, then was used for

115

immobilization of macromolecules via available for conjugation amino groups.24

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Briefly, the polyethylene frit was submerged in a solution containing 100 µL of 6.25 %

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GA in 0.1 M sodium phosphate buffer (pH 5.0) and incubated for 2 h at room

118

temperature. After washing with the same buffer, the goat-anti-mouse antibody (200

119

µL, at a concentration of 5 µg/mL) in carbonate buffer (0.1 M NaHCO3, 0.1 M

120

Na2CO3, pH 9.5), was adsorbed after incubation for 15 min at room temperature.

121

Then, PBS containing 1 % casein (500 µL per frit, 60 min incubation) was used for

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the blocking of free binding sites on the polyethylene frit surface.

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Development of the frit-based immunoassay

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The analysis procedure of the frit-based immunoassay includes sample passing,

125

tracer addition, washing to remove unbound tracer, and detection, and the analytical

126

signal was visible under UV light to the naked eye. (Figure S3) Briefly, two kinds of

127

MAbs were loaded onto the frit, and the column was incubated for 30 min, and excess

128

of unbound antibody was removed by washing with PBST. Subsequently, 2 mL of the

129

standard solution was passed through the frit on the test columns. Then, 200 µL of the

130

dual-color LQDs tracers were applied, and the frit on the test columns were incubated

131

for 6 min. To remove an excess of tracers, 5 mL of PBS with Tween 20 (0.05 %, w/v;

132

PBST) were passed through the test columns. Finally, the visual detection was

133

performed under UV light, and illustration of the results was show in Figure 1.

134

Sample pretreatment

135

Milk samples were purchased from local supermarkets, and were verified to be

136

AFM1 and PIR free by well-established LC-MS/MS methods.25, 26 Milk samples were

137

analyzed after a simple sample treatment outlined below.7 Briefly, the milk samples (5

138

mL) were transferred into 10 mL centrifuge tubes. Following the addition of 0.2 mL

139

of sodium nitroprusside (0.36 M) and 0.2 mL of zinc sulfate (1.04 M), the samples

140

were mixed for 1 min and then centrifuged at 3000 × g for 10 min at 4 °C. After

141

centrifugation, 2 mL of the supernatant was loaded on the test columns for the

142

detection of multiple chemical residues in duplicate with the developed immunoassay.

143

Analytical performance and real sample analysis

144

The analytical performances of the frit-based immunoassay were determined by

145

analyzing of the artificially-spiked samples by spiking the blank milk samples with 7



146

the appropriate standards. The cut-off values were defined as the lowest analyte

147

concentrations that did not provide luminescent signals in the test frits under UV

148

lights. Absence of luminescence analytical signal in the test frits was considered as a

149

positive result (concentration of analyte is above the cut-off level), while presence of

150

the analytical signal, independent on their brightness or saturation, was interpreted as

151

a negative result (concentration of analyte is below the cut-off level).

152

An intra-laboratory validation was performed using blank milk extracts

153

artificially spiked with analytes at concentrations less, equal, and above the

154

corresponding cut-off levels. The final validation of the frit-based immunoassay was

155

done using naturally contaminated milk samples and LC−MS/MS for confirmation.

156

Analytical characteristics were calculated according to Trullols et al.27 (Supporting

157

Information) and related information on the Commission Decision 2002/657/EC.

158

Results and discussion

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Microtiter plate-based multi-analyte ELISA

160

Prior to the development of frit-based immunoassays, single ELISA for AFM1

161

and PIR was performed, and the standard curves were shown in Figure S4. Here we

162

introduced the use of a mixture of two MAbs and their tracers, so that a global B/B0

163

measurement can be obtained in each analysis. The multianalyte ELISA system

164

employed a “cocktail” of analytes (AFM1 and PIR) and a “cocktail” of highly-specific

165

MAbs with negligible CR between them. As shown in Figure S5 and S6, a calibration

166

curve for only one analyte produced a sigmoidal curve with B/B0 ranging from 100%

167

to 50%. Therefore, the calibration curve of B/B0 for both analytes would produce a 8


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curve ranging from 100% to 0%. The mixed antibodies based ELISA system that

169

meets the requirements of a routine screening assay for the measurement of AFM1

170

and PIR in milk. When unknown samples are analyzed, the measurement of B/B0

171

values would provide the following information (Figure 2 and Figure S6):

172

Case 1: 0% < B/B0 < 50% indicated that the milk samples may be contaminated

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by both AFM1 and PIR simultaneously, and the residues of both analytes are higher

174

than the cut-off values.

175 176 177

Case 2: 50% < B/B0 < 100% indicated that the milk samples were contaminated by at least one analyte or perhaps more analytes at low concentration levels. Case 3: B/B0 ≈ 100% indicated that each analyte concentration is below its

178

cut-off values, and no contamination is measured.

179

Development of the frit-based immunoassay

180

Detection of low-molecular weight chemical analytes by antibody-based

181

immunoassay technique is sometimes hampered by the difficulties encountered in

182

immobilizing antibody or antigen on a solid support. In this paper, two different

183

specific antibodies, anti-AFM1, and anti-PIR, were co-immobilized onto the same

184

polyethylene frit, and the developed frit-based immunoassay format using red- and

185

green-emitting LQDs as a fluorescent labels. Pretreatment of the polyethylene frit

186

with certain doses of GA solution enhances its capacity for binding with

187

goat-anti-mouse antibody.28 If the GA-pre-treatment was omitted, the luminescent

188

intensities were nearly indistinguishable from the background signals, thus indicating

189

that the hapten was not immobilized. It is noteworthy that luminescent intensity 9



190

increases with increasing GA concentration, whereas luminescent intensity is

191

maximized at 6.25% GA. This pretreatment increases the specific signal in a

192

dose-dependent manner without augmenting the background or altering the specificity

193

of the assay.24, 29

194

In our previous work, the successful attempts for simultaneous measurement of

195

two analytes labeled with liposomes loaded with red- and green-emitting QDs were

196

performed. The goal of this study was to develop a dual-color (red- and

197

green-emitting QDs) quantum dot encoded frit-based immunoassay, which meets the

198

requirements of a routine screening assay, for the measurement of AFM1 and PIR in

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milk. The assay’s sensitivity is strongly influenced by the quantity of the specific

200

antibody (optimization of the quantity of the two antibodies was described in the

201

supporting information). No color development occurs when the quantity of target

202

analytes exceeds the available antibody binding sites. If there are no analytes in the

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sample, or the quantity of analytes is less than the corresponding number of antibody

204

binding sites, specific antibody binding sites would be available for binding respective

205

fluorescent labels. When unknown samples are analyzed, the luminescent intensity of

206

the frit would provide the following information (Figure 1):

207 208 209 210

Case 1: Orange fluorescence indicated that each analyte concentration is below its cut-off values, and no contamination is measured. Case 2: Red fluorescence indicated that the milk samples were contaminated by PIR (higher than the cut-off value).

10


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Case 3: Green fluorescence indicated that the milk samples were contaminated by AFM1 (higher than the cut-off value).

213

Case 4: No fluorescence indicated that the milk samples may be contaminated by

214

both AFM1 and PIR simultaneously, and the residues of both analytes are higher than

215

cut-off values. Confirmatory methods would be needed to provide the final decision

216

of the exact residue and whether the residue levels are acceptable for consumption.

217

Sample pretreatment

218

Milk is a very complicated matrix: it contains many macromolecular substances

219

(proteins, sugars and lipids, among others). In this paper, the diluted milk matrix and

220

deproteinated milk matrix were tested by the developed frit-based immunoassays, and

221

the results were shown in Figure 3b and Figure 3c, respectively. The macromolecular

222

substances could be retained on the frit, and decreased the luminance signals that

223

could lead to false results. Thus, sodium nitroprusside and zinc sulfate were added to

224

remove these substances via precipitation. After de-proteination, the matrix

225

interference was reduced to an insignificant level, and the supernatant could be

226

directly used on the frit-based immunoassay for testing.

227

Analytical performance

228

A series of tests with AFM1 and PIR standard solutions and spiked milk samples

229

were conducted using the developed assay, and the brightness of the luminance

230

intensity decreased with respect to increasing concentrations on the calibration curves

231

in milk. The obtained visual results for the AFM1 and PIR are presented in Figure 3c.

232

The results indicated that the luminescent intensity was inversely proportional to the 11



233

analyte concentration. The frit-based immunoassays were characterized with a cut-off

234

value of 0.02 ng/mL for AFM1 and 0.5 ng/mL for PIR, bearing in mind the extraction

235

and dilution of samples. Both cut-off values of the frit-based immunoassay satisfied

236

the requirements of the MRLs set by China.

237

Milk samples were spiked with AFM1 and PIR at three concentration levels. The

238

final AFM1 concentrations were 0.02, 0.1, and 0.5 ng/mL, and the final PIR

239

concentrations were 0.5, 2.5, and 10 ng/mL. The spiked milk samples were tested by

240

the developed frit-based immunoassay. False positive, false negative, specificity, and

241

sensitivity rates of assay were calculated according a contingency table using a

242

two-category classification (Table S2). As depicted in Table 1, the frit-based

243

immunoassay was able to correctly analyze milk contaminated with AFM1 or PIR or

244

with both over the cut-off values. The rates for false positive and false negative results

245

were both below 5%, and the specificity and sensitivity rates were >95%, which fulfill

246

the requirements set by the Commission Decision 2002/657/EC for a screening

247

method30-32, i.e., only those analytical techniques, for which it can be demonstrated in

248

a documented, traceable manner that they are validated and have a false compliance

249

rate of < 5 % at the level of interest shall be used for screening purposes.

250

Assay Validation

251

The reliability of this immunoassay was demonstrated by blind analysis of spiked

252

samples prepared at the China National Center for Food Safety Risk Assessment

253

(Beijing, China). Ten milk samples, artificially spiked with AFM1 and PIR standards,

254

were tested by the frit-based immunoassays, and further confirmed by 12


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well-established LC-MS/MS methods33, 34. From the ten milk samples analyzed, no

256

false negative results were obtained for spiked samples (Table 2). Furthermore, five

257

milk samples artificially spiked with both analytes were used for the final validation,

258

and the comparison of the immunoassays and chromatography results showed good

259

agreement for both negative and positive samples (Table 3).

260

Comparison with previous reported immunoassay formats

261

During the last two decades, various kinds of immunochemical assays were

262

developed for detection of low-molecular weight chemicals residues in foods.

263

However, traditional micro-titer plate-based immunoassays usually use enzymes as

264

labels, and often focus on the detection of a single analyte. The novel frit-based

265

immunoassay allows one to combine in a single procedure and in one column the

266

preconcentration onto the immunoaffinity polyethylene frits and the detection of the

267

target analytes. Comparison between the frit-based immunoassay and ELISA method

268

were described in Table 4. In the term of methodology itself, the frit-based

269

immunoassay has its unique advantages. First, the frit-based immunoassay has a

270

broad loading volume (from 1 to 10 times column bed), which could improve the

271

sensitivity of detection effectively, while the loading volume of ELISA is usually from

272

50-100 µL.35 Second, the operation is simple and rapid, and naked eye could be used

273

for judgment. These advantages indicated that the frit-based immunoassay could also

274

act as effective screening methods for food safety surveillance purpose.

275

This new approach could offer a significant reduction in cost per analysis since

276

the frits are much cheaper than previously reported alternatives. Immobilizing 13



277

antibodies on polyethylene frits offers several advantages, such as the simplicity of

278

antibody coupling onto the surface of the frits, and the possibility of long-term storage

279

of the coupled frits under refrigeration conditions. In addition, the results of the

280

developed technique could be directly observed under UV light, which makes the

281

no-instrumental method suitable for rapid screening purposes in the field. The

282

liposome-loaded with dual-color QDs were evaluated as labels in the development of

283

multiplex immunoassays. In contrast to previous research, encapsulation of QDs into

284

liposomes minimized nonspecific absorption, facilitated bioconjugation, and

285

increased luminescent intensity. Besides, the obtained LQDs were not prone to any

286

non-specific interaction with the surface of the polyethylene frit. The frit-based

287

immunoassay combined preconcentration of target analytes in food matrices, resulting

288

in higher sensitivity. It was shown that the novel frit-based immunoassay led to an

289

approximately 10-fold increase in immunoassay sensitivity, compared with traditional

290

ELISAs (according to data presented in our previous work3, 8). In addition, the whole

291

test could be performed within 30 min, which greatly shortens the required assay

292

duration.

293

In this paper, detailed information on developing rapid and sensitive frit-based

294

immunoassays was described for on-site screening multiple analyte residues in milk.

295

The novel frit-based immunoassay allows one to combine dual-color QD (QD540 and

296

QD630) labels in one-column preconcentration onto the frits for the detection of

297

AFM1 and PIR. The application of QDs facilitates frit-based immunoassays, and

298

therefore can contribute to enhanced throughput and reduced analysis time. 14


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ASSOCIATED CONTENT Supporting information for additional experimental section details is available

300 301

free of charge via the internet at http://pubs.acs.org.

302

303

Corresponding Author

AUTHOR INFORMATION

∗ E-mail: [email protected] (X. Wang); Tel: +86-755-8667-1936; Fax:

304 305

+86-755-8667-1906.

306

Funding

307

This work was supported by National Natural Science Foundation of China (No.

308

31602103), Natural Science Foundation of Guangdong Province, China (No.

309

2014A030310289), Science and Technology Planning Project of Guangdong Province,

310

China (No. 2016A020210055), Medical Scientific Research Foundation of

311

Guangdong Province, China (No. A2016078), Shenzhen Basic Research Project (No.

312

JCYJ20160307114724751 and JCYJ20150324141711558) and Natural Science

313

Foundation of SZU (No. 201576).

314

Notes

315

The authors declare no competing financial interest.

316

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317

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He, J.; Wu, N.; Luo, P.; Guo, P.; Qu, J.; Zhang, S.; Zou, X.; Wu, F.; Xie, H.; Wang, C.;

Wang, X.; Luo, P.; Chen, J.; Huang, Y.; Jiang, W., Development of a quantitative

Jiang, W.; Beier, R. C.; Luo, P.; Zhai, P.; Wu, N.; Lin, G.; Wang, X.; Xu, G., Analysis of

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determination of cereal-borne mycotoxins. Talanta 2016, 160, 66-71.

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15. Petryayeva, E.; Algar, W. R.; Krull, U. J., Adapting fluorescence resonance energy

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transfer with quantum dot donors for solid-phase hybridization assays in microtiter plate

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format. Langmuir 2013, 29, 977-987.

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16. Hou, J.-Y.; Liu, T.-C.; Ren, Z.-Q.; Chen, M.-J.; Lin, G.-F.; Wu, Y.-S., Magnetic

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particle-based time-resolved fluoroimmunoassay for the simultaneous determination of

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α-fetoprotein and the free β-subunit of human chorionic gonadotropin. The Analyst 2013, 138,

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3697-3704.

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17. Ranzoni, A.; den Hamer, A.; Karoli, T.; Buechler, J.; Cooper, M. A., Improved

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immunoassay sensitivity in serum as a result of polymer-entrapped quantum dots: ‘papaya

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particles’. Analytical Chemistry 2015, 87, 6150-6157.

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18. Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I.

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L.; Mattoussi, H., Multiplexed toxin analysis using four colors of quantum dot fluororeagents.

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Analytical Chemistry 2003, 76, 684-688.

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19. Goryacheva, I. Y.; Speranskaya, E. S.; Goftman, V. V.; Tang, D.; De Saeger, S.,

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Synthesis and bioanalytical applications of nanostructures multiloaded with quantum dots.

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20. Jiang, W.; Zhang, H.; Li, X.; Liu, X.; Zhang, S.; Shi, W.; Shen, J.; Wang, Z.,

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Monoclonal antibody production and the development of an indirect competitive

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enzyme-linked immunosorbent assay for screening spiramycin in milk. Journal of

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Agricultural and Food Chemistry 2013, 61, 10925-31.

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21. Beloglazova, N. V.; Speranskaya, E. S.; De Saeger, S.; Hens, Z.; Abe, S.; Goryacheva, I.

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Y., Quantum dot based rapid tests for zearalenone detection. Analytical and Bioanalytical

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Chemistry 2012, 403, 3013-24.

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Knopp, D.; Niessner, R.; De Saeger, S.; Goryacheva, I. Y., Quantum dot loaded liposomes as

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fluorescent labels for immunoassay. Analytical Chemistry 2013, 85, 7197-7204.

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polyethylene for protein immobilization. Biomaterials 1993, 14, 817-822.

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terbutryn using direct hapten linkage to a glutaraldehyde network on the polystyrene surface

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milk by UHPLC–MS/MS. Food Chemistrry 2014, 146, 242-249.

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27. Trullols, E.; Ruisánchez, I.; Rius, F. X., Validation of qualitative analytical methods.

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D., Glutaraldehyde (GA)-hapten adducts, but without a carrier protein, for use in a specificity

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antibody (AHA-2) as a model. Journal of Biochemistry 1999, 126, 1170-4.

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of heterology in a monoclonal antibody-based ELISA utilizing direct hapten linkage to

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polystyrene microtiter plates. Journal of Immunological Methods 2005, 304, 68-77.

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30. Commission of the European Communities. Commission Decision (EC) No. 657/2002 of

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12 August 2002 implementing Council Directive 96/23/EC concerning the performance of

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analytical methods and the interpretation of results. Official Journal of the European

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Communities 2002, L221, 8.

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31. Jiang, W.; Beier, R. C.; Wang, Z.; Wu, Y.; Shen, J., Simultaneous screening analysis of

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3-methyl-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid residues in edible

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animal tissues by a competitive indirect immunoassay. Journal of Agricultural and Food

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Chemistry 2013, 61, 10018-25.

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Competitive

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Phenylethanolamine A Residues in Pork Samples. Food Analytical Methods 2016, 9,

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National food safety standard: Determination of aflatoxin M1 in milk and milk products.

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GB5413.37–2010, 2010.

424

34. General Administration of Quality Supervision, Inspection and Quarantine of the

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People’s Republic of China. Determination of spiramycin, pirlimycin, oleandomycin,

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tilmicosin, erythromycin and tylosin residues in milk and milk powder: LC–MS/MS method.

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GB/T 22988–2008, 2008.

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35. Xu, F.; Jiang, W.; Zhou, J.; Wen, K.; Wang, Z.; Jiang, H.; Ding, S., Production of

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monoclonal antibody and development of a new immunoassay for apramycin in food. Journal

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of Agricultural and Food Chemistry 2014, 62, 3108-3113.

Indirect

Enzyme-Linked

Immunosorbent

431

18


Assay

for

Screening

Page 19 of 27


432

Figure Captions

433

Figure 1. Principles of dual-color QDs encoded frit based immunoassay for the

434

simultaneous screening of AFM1 and PIR residues in milk.

435

Figure 2. The dose-response standard curve and the schematic representation of

436

the competitive ELISA procedure. (a) AFM1 is the reference analyte in the

437

multianalyte ELISA, (b) PIR is the reference analyte in the multianalyte ELISA,

438

and (c) represents that the multianalyte ELISA used a “cocktail solution”

439

(AFM1 and PIR) as the reference analytes.

440

Figure 3. The detection capabilities of the developed frit-based immunoassay

441

for detecting AFM1 and PIR with dual-color liposome loaded with QDs.

442

19



Page 20 of 27

443

Table 1. Analytical performance of the frit-based immunoassay for AFM1 and

444

PIR residue analysis. Parameters

AFM1

PIR

Total number of tests (N)

240

240

Cut-off value (ng/mL)

0.02

0.5

False-positive rate, % (Nfalse positive/N−) × 100)

4.2

2.5

False-negative rate, % (Nfalse negative/N+) × 100)

3.3

1.7

Specificity rate, % (Nnegative/N−) × 100)

95.8

97.5

Sensitivity rate, % (Npositive/N+) × 100)

96.7

98.3

445 446

20


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447

Table 2. Comparison of AFM1 and PIR analyses using LC–MS/MS and the

448

frit-based immunoassay in blind milk samples. Analyte

Sample No.

LC–MS/MS (ng/mL)

Frit-based immunoassaya (n = 4)

AFM1

A1

0.008

– – – – –a

A2

0.15

+ + + + +a

A3

0.26

+++++

A4

1.6

+++++

A5

5.2

+++++

B1

0.05

–––––

B2

1.6

+++++

B3

3.1

+++++

B4

10.2

+++++

B5

20.9

+++++

PIR

449

a

–, absence of analyte; +, presence of analyte.

450

21



Page 22 of 27

451

Table 3. Comparison of AFM1 and PIR analyses using LC-MS/MS and the

452

frit-based immunoassay in blind milk samples. Frit-based immunoassaya (ng/mL)

LC-MS/MS Analyte

AFM1 + PIR

453

a

Sample No.

(ng/mL) AFM1

PIR

AFM1

PIR

M1

0.008

0.05

– – – – –a

–––––

M2

0.015

0.09

–––

––

M3

0.15

1.6

+ + + + +a

+++++

M4

0.58

5.2

+++++

+++++

M5

1.5

10.8

+++++

+++++

–, absence of analyte; +, presence of analyte.

454

22


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455

Table 4. The comparison between frit-based immunoassay and ELISA.

Parameters

Frit-based immunoassay

Immuo-reaction carrier polyethylene frit

ELISA polystyrene micro-plate well

pre-concentration,

specific specific interaction between

Principles

interaction between antibody antibody and antigen and antigen quantitative

Results assessment

or

qualitative semi-quantitative

Instrument no

yes

requirement simple, suitable for on-site complicated,

suitable

Sample treatment

Sample

screening

laboratory analysis

Na mL (N>1)

50 µL - 100 µL

concentration 20Na times

dilution 1 - 10 times

15 - 30 min

90 - 120 min

loading

volumes Dilution/concentration factor Total detection time 456

a

the loading volume of the samples.

457

23


for


458

459 460 461

Figure 1. Principles of dual-color QDs encoded frit based immunoassay for the

462

simultaneous screening of AFM1 and PIR residues in milk.

463

24


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Page 25 of 27


464

100

100

90

90

80

80

B/B 0 (% )

B/B0 (%)

B/B 0 (% )

100

60

80

70

40

70

60

60

50

50

0.01

0.1

1

AFM1 (ng/mL)

10

20 0.1

1

10

PIR (ng/mL)

100

0

AFM1 0.01 PIR 0.1

0.1

1

10

1

10

100

"Cocktail" analyte (ng/mL)

465 466 467

Figure 2. The dose-response standard curve and the schematic representation of the

468

competitive ELISA procedure. (a) AFM1 is the reference analyte in the multianalyte

469

ELISA, (b) PIR is the reference analyte in the multianalyte ELISA, and (c) represents

470

that the multianalyte ELISA used a “cocktail solution” (AFM1 and PIR) as the

471

reference analytes.

472

25



473

474 475 476

Figure 3. The detection capabilities of the developed frit-based immunoassay for

477

detecting AFM1 and PIR with dual-color liposome loaded with QDs.

478

26


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479

For TOC only

480 481

27


A Dual-Color Quantum Dots Encoded Frit-Based Immunoassay for

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