(Centrifugation) and Qualitative (Filtration) Immunoassays - American

Sep 30, 2016 - ABSTRACT: Current methods to detect gentamicin (GEN), a broad-spectrum antibiotic that causes ototoxicity and nephrotoxicity when prese...
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Use of Carbon Nanotubes as a Solid Support to Establish Quantitative (centrifugation) and Qualitative (filtration) Immunoassays to Detect Gentamicin Contamination in Commercial Milk Kun Zeng, Wei Wei, Ling Jiang, Fang Zhu, and Daolin Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03332 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 2, 2016

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Journal of Agricultural and Food Chemistry

Use of Carbon Nanotubes as a Solid Support to Establish Quantitative (centrifugation) and Qualitative (filtration) Immunoassays to Detect Gentamicin Contamination in Commercial Milk

Kun Zeng1,2#, Wei Wei1,2#, Ling Jiang1,2, Fang Zhu1, Daolin Du1,2*

1

School of the Environment and Safety Engineering, Jiangsu University, 301 Xuefu Road,

Zhenjiang, Jiangsu 212013, China 2

Institute of Environment and Ecology, Jiangsu University, 301 Xuefu Road, Zhenjiang

212013, China

#

These authors contributed equally to this work and should be considered co-first authors.

* To whom enquiries should be addressed Email: [email protected] Telephone: 0511-88780955 Keywords: Gentamicin; MWCNTs; Filtration

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ABSTRACT:

2

Current methods to detect gentamicin (GEN), a broad-spectrum antibiotic that causes

3

ototoxicity and nephrotoxicity when present in excess, have several limitations. Hence, we

4

have developed two methods using multi-walled carbon nanotubes (MWCNTs), as a solid

5

support, to detect GEN. Hybridoma cells (2D12) producing high sensitivity antibodies

6

against GEN were established. The goat anti-mouse antibody (GAM) was immobilized on

7

MWCNTs directly or using bifunctional PEG as a linker. Based on the physical

8

characteristics of MWCNTs, a quantitative method involving centrifugation separation and a

9

qualitative method involving filtration separation were established. Various experimental

10

parameters were optimized for GEN detection and recovery tests were performed. For the

11

quantitative method, the limit of detection (LOD) was 0.048 ng/mL whereas for the

12

qualitative method, a LOD of 0.1 ng/mL was observed by the naked eye. The proposed

13

immunoassays were applied to commercial milk samples. Thus, these methods show

14

potential application for the detection of GEN.

15 16 17 18

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1. INTRODUCTION

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Gentamicin (GEN), as one of the most widely used aminoglycoside antibiotics, exhibits

21

broad-spectrum bactericidal action against both gram-negative and gram-positive bacterial

22

infections in both humans and livestock1. Excessive levels of GEN in food of animal origin

23

can have adverse effects on human health such as increasing the incidences of ototoxicity and

24

nephrotoxicity2. According to the European Agency for the Evaluation of Medical Products,

25

the maximum residue limits (MRLs) for gentamicin in milk have been set to be 100 ng/mL3.

26

Conventional analytical methods for the detection of gentamicin mainly include

27

high-performance liquid chromatography (HPLC)

28

spectrometry (LC-MS)5,7, microbiological assays8, and immunoassays1,9,10,11. Generally,

29

HPLC and LC-MS methods are expensive and require a specialized operator while

30

microbiological assays have poor sensitivity and are time-consuming. Therefore,

31

immunoassays that are highly sensitive and that can be adapted to high throughput formats

32

have attracted the attention of analytical chemists.

4,5,6

, liquid chromatography-mass

33

Immunoassays can be of either a heterogeneous or a homogeneous type. In

34

heterogeneous immunoassays, antibodies/antigens are fixed to a solid support. Following

35

capture of the antigen by the immobilized antibody (or vice-versa), non-bound reagents can

36

be easily removed from the solid support by a physical separation methodology. In

37

homogeneous immunoassays, antibodies and antigens interact in solution and do not require

38

an additional separation step. Heterogeneous immunoassays generally demonstrate higher

39

sensitivity compared to homogeneous immunoassays, so the former have become the most

40

popular type of immunoassay. In heterogeneous immunoassays, the solid supports have

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different physical and chemical properties, all of which influence assay performance.

42

Microtiter plates, made of polystyrene or polyvinyl chloride, are the most widely used solid

43

support; they demonstrate high protein adsorption and small well-to-well variation. When the

44

antibody attaches to the planar surface by adsorption, the avidity of antibody is lowered and

45

consequently more time is required to achieve the antibody-antigen binding equilibrium; this

46

can influence the performance of assays, such as sensitivity. Recently, a new generation of

47

materials has been adopted as solid support in immunoassays, including magnetic particles

48

(MNPs)12,13,14, metal nanoparticles15,16,17, and carbon nanotubes (CNTs)18,19,20.

49

CNTs were first discovered in Japan in 1991 and are formed from sp2 bonded carbon

50

atoms rolled up into the shape of a tubular structure21. CNTs include single-walled (SWCNTs)

51

and multi-walled (MWCNTs). SWCNTs consist of one layer of cylindrical graphene with a

52

diameter between 0.4 and 2 nm whereas MWCNTs contain several concentric graphene

53

sheets with diameters between 2 and 100 nm22. Due to the unique mechanical, electrical, and

54

thermal properties of CNTs23, CNTs have attracted the attention of scientists for nearly two

55

decades and have a wide range of uses in the biomedical field, including uses as

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drug-delivery carriers24, gene delivery systems25 and immunodetection methods26. In

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analytical processes, because of their advantage of having a large surface area, CNTs can be

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used as solid supports to carry proteins, nucleic acids, and drugs either through covalent or

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non-covalent binding. Currently CNTs have principally been used in three fields: a)

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electrochemical biosensors: CNTs can mediate electron-transfer reactions between

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electroactive species and can thus be developed as electrical sensors19,27; b) signal

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amplification:CNTs can be linked with enzymes or agents, such as other nanoparticles, by

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exploiting the high absorption capacity of CNTs26,28;or c) as a versatile label: CNTs can be

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used as signaling molecules replacing gold nanoparticles in the lateral flow assay, producing

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the characteristic black bands for visual detection20,29 However, CNT based bioassays suffer

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from the requirement for numerous, tedious steps, and a requirement for sophisticated

67

instrumentation.

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In this study, we aimed to develop simple, sensitive, and user-friendly immunoassays to

69

detect GEN using MWCNTs as a solid support and to demonstrate the effectiveness of such

70

assays to detect GEN in samples of commercially available milk.

71 72

2. MATERIALS AND METHODS

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2.1. Reagents and materials

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Gentamicin sulfate, tetramethylbenzidine (TMB), 1-ethyl-3-(3-dimethylamino-propyl)

75

carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Freund’s complete

76

adjuvant (FCA), Freund’s incomplete adjuvant (FIA), bovine serum albumin (BSA),

77

ovalbumin (OVA), goat anti-mouse antibody (GAM), NH2-PEG-COOH and gelatin were

78

purchased from Sigma-Aldrich (St. Louis, MO, USA). MWCNTs (Product Number:

79

TNSMC8) were purchased from Chengdu Organic Chemicals Co. Ltd (China). 0.22 µm

80

nitrocellulose (NC) membranes were from Sinopharm Chemical Reagent Beijing Co., Ltd

81

(China). ImmunoPure® Monoclonal Antibody Isotyping Kit were purchased from Pierce

82

(Rockford, IL). ELISA plates were from Costar (Cambridge, MA, USA). Absorbance

83

measurements were made with a microplate reader (BioTek Instruments, Inc. Winooski, VT).

84

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2.2 Preparation of gentamicin-protein conjugates

86

To prepare the immunogen (BSA-GEN), GEN (30 mg) dissolved in 5 mL PBS, was

87

mixed with 10 mg BSA. EDC (50 mg), dissolved in 2 mL PBS, was then added by dropping

88

it into the above solution. The mixture was stirred for 2 h at 4°C and dialyzed against PBS for

89

three days. The solution was then aliquoted and stored at -20°C. To prepare coating

90

antigens(OVA-GEN), 10 mg GEN, 10 mg OVA and 50 mg EDC were dissolved in 6 mL PBS.

91

The reaction process and subsequent storage of samples was as described above. In order to

92

conjugate gentamicin with HRP, 5 mg HRP was added to 5 mg GEN in 1 mL PBS. EDC (10

93

mg) dissolved in 2 mL PBS was then dropped into the solution. The reaction process and

94

subsequent storage of samples was also as described above.

95 96

2.3 Development of anti-gentamicin MAb

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BSA-GEN was emulsified in FCA and BALB/c female mice (18-22 g) were immunized

98

subcutaneously at a dose of 100 µg per mouse. After four weeks, mice were injected with 50

99

µg BSA-GEN in FIA and three boosts of immunogen were then administered every two

100

weeks until the fifth immunization. Blood samples were collected from the mouse tail and

101

assessed for immunoreactivity against GEN via ELISA. The cell fusion and hybridoma

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screening methods were developed according to Zeng30. Antibody class and subclass

103

determinations were performed according to the operation guide in the ImmunoPure®

104

Monoclonal Antibody Isotyping Kit. Ascitic fluids were produced in mineral oil-primed

105

BALB/c mice. MAbs were purified from the ascitic fluid by caprylic acid-ammonium sulfate

106

precipitation.

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2.4 Indirect competitive ELISA for GEN

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An indirect competition ELISA format was utilized to measure GEN binding and

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cross-reactivity to related compounds. First, 96-well plates were coated with OVA-GEN

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diluted in carbonate buffer (0.85 mol/L, pH 9.6) by incubation at 4 °C overnight. Then plates

112

were then washed once with PBST and blocked with 200 µL PBS containing 1% gelatin at

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37 °C for 2 h. Then, 50 µL GEN standards, followed by 50 µL anti-GEN antibody, was added

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to the wells and incubated at 37 °C for 1 h. After three washes with PBST, 100 µL IgG-HRP

115

(1:5000) was added and reacted for 30 min at 37 °C. After three washes with PBST, the

116

peroxidase activity was revealed with freshly prepared TMB substrate solution, and the

117

enzymatic reaction was stopped after 10 min by adding 50 µL H2SOS4 (2 mol/L). The

118

absorbance was immediately read at 450 nm with a reference wavelength of 630 nm.

119 120

2.5 Preparation of MWCNTs-GAM and MWCNTs-PEG-GAM

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To obtain carboxylated MWCNTs, MWCNTs were treated with a 3:1 (v/v) mixture of

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sulfuric and nitric acids at 65°C for 4 h. The carboxylated MWCNTs were filtered and

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washed repeatedly with distilled water until the pH of water was neutral. The product was

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dried at 50°C

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Carboxylated MWCNTs (1 mg) were uniformly dispersed in 2 mL ddH2O using

126

ultrasound for 1 min. NHS (50 mg) and EDC (5 mg) dissolved in 1 mL MES (0.5 mol/L, pH

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6.1) were added to the MWCNTs solution and stirred for 30 min at room temperature. After

128

centrifugation at 13000 rpm for 10 min, the supernatant was discarded and the precipitate was

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washed with MES (50 mmol/L, pH 6.1) to remove unconjugated EDC or NHS. After washing

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twice, the precipitate was resuspended in 1 mL MES (50 mmol/L, pH 6.1) and 400 µg GAM

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was added. The mixture was stirred overnight at 4°C. The conjugated MWCNTs-GAM was

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washed three times using PBS (0.01 mol/L, pH 7.2) and stored in 1 mL PBS at 4°C.

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Carboxylated MWCNTs (1 mg), dispersed in 1 mL ddH2O, were mixed with 12.4 mg

134

EDC, 7.2 mg NHS and 2 mg NH2-PEG-COOH. The solution was stirred for 2 h at room

135

temperature. The process for washing and binding of GAM was as described above.

136 137

2.6 Quantitative assay using centrifugation

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In a 1.5-mL tube, 2.5 µL MWCNTs-PEG-GAM was dispersed into 50 µL PBS (0.01

139

mol/L) containing 2% BSA. Then, 50 µL anti-GEN MAb, 50 µL HRP-GEN, and 50 µL GEN

140

standard were added to the suspension in turn. The mixture was vortexed and incubated at

141

37°C for 30 min. After centrifugation at 13000 rpm for 10 min, the supernatant was discarded

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and the precipitate was washed three times with PBST. Bound HRP was detected by adding

143

freshly prepared TMB solution to the precipitate and color development was allowed to

144

proceed. Fifteen minutes later, the reaction was stopped by adding 50 µL H2SO4 (2 mol/L).

145

The supernatant (200 µL) was added to the well of a 96-well plate and the absorbance was

146

immediately read at dual wavelengths of 450 nm and 630 nm.

147 148

2.7 Qualitative assay by membrane filtration

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NC membranes were blocked with 1% BSA for 2 h at 25°C and then dried at room

150

temperature. Following the incubation of MWCNTs-PEG-GAM, anti-GEN MAb, HRP-GEN

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and GEN standard as described above, the mixture was filtered through the NC membrane

152

and the membrane was washed with 5 mL PBST. TMB solution (200 µL) was applied to the

153

membrane using a pipette and color development was recorded using a digital camera.

154 155

2.8 Optimization of parameters

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To minimize non-specific binding, PBS containing different proteins as blocking agents

157

was tested. BSA (1–5%), casein (1–5%) and gelatin (1–5%) were tested respectively and

158

background absorbance was subtracted. To maximize the immobilization of anti-GEN MAb,

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differing amounts of GAM (50–1600 µg) were mixed with MWCNTs-PEG (1 mg). Then,

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anti-GEN

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MWCNTs-PEG-GAM and the absorbance was read at dual wavelengths of 450 nm and 630

162

nm. To optimize the dilution of the antibody and the HRP-conjugate, different combinations

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of anti-GEN antibody (1:500 and 1:2000) and HRP-GEN (1:1000, 1:4000 and 1:8000) were

164

tested The maximum absorbance (ODmax) and background were recorded and the IC50 was

165

calculated, where IC50 is the concentration at which 50% of the antibodies are bound to the

166

analyte.

MAb

(1:4000)

and

HRP-GEN

(1:2000)

were

mixed

with

the

167

The processes used for optimization of pH and reaction time were as follows. GEN was

168

diluted in a range of 0.01 mol/L PBS having different pH values, namely, 5.7, 6.2, 6.8, 7.4,

169

and 8.0, respectively. MWCNTs-PEG-GAM, anti-GEN MAb, HRP-GEN and GEN standard

170

were mixed together for 5 min, 15 min, 30 min, 45 min and 60 min respectively. ODmax and

171

IC50 were determined.

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2.9 Analysis of commercially available milk

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Five samples of commercially available milk were obtained from local markets

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(Zhenjiang, China). To reduce any potential interference of milk ingredients, samples were

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serially diluted with PBS before analysis.

177 178

3. RESULTS AND DISCUSSION

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3.1 Characterization of anti-GEN MAbs

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Through the process of five mice immunizations, one mouse, which produced antibodies

181

with high titer and sensitivity, was chosen for the preparation of the hybridoma. Following

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three rounds of subcloning, one hybridoma cell, (2D12) producing high sensitivity antibodies,

183

was successfully obtained. The antibody was identified to be of the IgG2a subclass with a

184

kappa light chain.

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The cross-reactivity of 2D12 with structural analogs of GEN was examined in order to

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understand its selectivity (Table 1). The antibody 2D12 showed only weak cross reactivity

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with tobramycin (0.31%) and had no cross reactivity with neomycin, kanamycin, ribose

188

neomycin and amikacin. Using the antibody 2D12, an indirect competitive ELISA was

189

developed. Based on the optimized concentration of

190

anti-GEN antibody (10 mg/mL, 1:50000), the IC50 was calculated as 0.095 ng/mL, which was

191

lower than the value previously obtained by Wei et al.9, Chen et al.1, and Shalev et al.31, who

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obtained IC50 values of 0.95 ng/mL, 0.92 ng/mL, and 130 µg/mL, respectively. The limit of

193

detection (LOD), defined as the concentration giving a 10% decline of the signal at zero

194

binding (i.e., maximal signal) was 0.026 ng/mL, and the linear range was 0.042–0.2438

OVA-GEN (0.5 µg/mL) and

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Journal of Agricultural and Food Chemistry

ng/mL.

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3.2 Characterization of MWCNTs and covalently immobilized antibodies

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In this study, MWCNTs were chosen as a solid support for covalent immobilization of

199

GAM, because they provide a large surface area that can maximize the amount of

200

immobilized antibody. The GAM was attached to the surface of the MWCNTs by one of two

201

methods both of which take advantage of the carboxyl moiety that can be easily introduced

202

into MWCNTs by acid treatment. In one method, GAM was bound directly to the surface of

203

the MWCNTs using EDC/NHS. In the second method, bifunctional PEG (NH2-PEG-COOH)

204

was used as a linker between GAM and the MWCNTs. In previous studies PEG has proven to

205

be a effective spacer to maintain the steric structure of the antibody near surfaces32. Fig. 1

206

shows transmission electron microscopy (TEM) images of MWCNTs (carboxylated or

207

non-carboxylated) before and after antibody conjugation. No significant morphological

208

changes were observed when comparing MWCNTs and carboxylated MWCNTs. However, it

209

was noted that carboxylated MWCNTs dispersed uniformly in water by ultrasound better than

210

non-carboxylated MWCNTs. For both MWCNTs-GAM and MWCNTs-PEG-GAM, it was

211

observed that the nanotube pore size decreased significantly, while the surface showed

212

increased roughness suggesting that the antibody had been successfully conjugated with

213

MWCNTs.

214 215 216

3.3 Blocking of MWCNTs Minimization of nonspecific binding (NSB) is crucial for establishing sensitive and

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reproducible analytical methods33. To avoid NSB to MWCNTs, blocking solutions containing

218

different proteins at various concentrations were used to attempt to reduce the background.

219

As shown in Fig. 2, for MWCNTs-GAM, regardless of the blocking solution used, a high

220

background was noted. In complete contrast, the background was decreased significantly for

221

MWCNTs-PEG-GAM. Based on this it appears that PEG plays an important role in antibody

222

immobilization with respect to NSB. In particular, PEG may help to maintain the steric

223

structure of antibody and so prevent loss of its activity32. On the other hand, when antibody is

224

linked to MWCNTs directly, there are potentially many gaps on the surface due to the large

225

size of the antibody molecule, which could lead to greater non-specific absorption of proteins,

226

particularly HRP, and so contribute to the high background. However when PEG is

227

introduced, it provides an improved steric space so that small sized blocking proteins can

228

easily

229

MWCNTs-PEG-GAM could be optimized by using different blocking solutions. The

230

background was found to be lowest using either a 2% or 5% BSA solution. Therefore a 2%

231

BSA solution was chosen as the blocking solution.

occupy

the

non-specific

sites.

The

reduction

in

background

using

232 233

3.4 Quantitative assay using centrifugation

234

For heterogeneous immunoassays, quantitative signals are obtained by reporters fixed on

235

a solid support. Many materials can be used as a solid support in the immunoassay field,

236

including MNPs, CNTs, and others. Different separation methods are adopted according to

237

the characteristic of the carriers. With a magnetic core coated with a shell, MNPs can be

238

separated and enriched by magnetic field with high separation efficiency and can be used as

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carriers to load bioactive molecules due to be high surface area to volume ratio, and this has

240

become a versatile tool in the biotechnology field12,13,14. However, magnetic instrumentation

241

is needed to separate the bound and unbound reagents in this type of immunoassay. Because

242

of cost, however, the popularity of this method is limited. In this study, following incubation

243

of MWCNTs-PEG-GAM, anti-GEN MAb, GEN standard and HRP-GEN, centrifugation was

244

employed as a means to separate the immobilized HRP-GEN from any unbound soluble

245

HRP-GEN.

246

To maximize the assay signal, the amount of GAM used was optimized (Fig. 3a). The

247

absorbance rapidly increased from 50 µg to 400 µg and plateaued at 400 µg, signifying that

248

the MWCNTs had bound the maximum amount of antibody possible. Therefore, the amount

249

of GAM was set at 400 µg. To determine the optimal concentration of anti-GEN antibody and

250

HRP-GEN, different combinations of each were tested (Table 2). A high background was

251

observed at a 1:2000 dilution of HRP-GEN but the background was reduced significantly at

252

lower dilutions. Based on ODmax and IC50, the anti-GEN antibody and HRP-GEN were used

253

at dilutions of 1:2000 and 1:4000 respectively. To identify the optimal antigen-antibody

254

equilibration time, the effect of changing the mixing time over a range from 5–60 min was

255

examined (Fig. 3b). The absorbance signal rapidly increased from 5 to 30 min and plateaued

256

at times greater than 30 min. The IC50 value decreased from incubation time of 5 to 30 min

257

and increased with incubation times from 30 to 60 min. Based on these data the optimum

258

interaction time was considered as 30 min. As shown in Fig. 3c, the absorbance signal

259

gradually increased from pH 5.7 to 6.8 and essentially plateaued at pH values greater than 6.8.

260

The IC50 value decreased from pH 5.7 to pH 7.4 with the lowest value being observed at pH

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7.4. Based on these data the optimum pH was chosen to be 7.4.

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Fig. 4 shows a calibration curve using the centrifugation separation method. The IC50

263

value was calculated as 0.195 ng/m and the LOD was 0.048 ng/mL, which indicated higher

264

sensitivity compared with previous reports1,

265

0.080–0.512 ng/mL.

9, 31

. The linear range of the assay was

266 267

3.5 Qualitative method by filtration separation

268

Visualization assays based on membranes, such as the lateral flow assay (LFA) and dot

269

ELISA, have been widely used in clinical diagnosis, food safety and environmental

270

analysis15,16.17.34,35. Antibodies or antigens are immobilized on NC or PVDF membranes and

271

the color of a line or a dot are developed by different molecular signal generators, such as

272

gold nanoparticles, latex particles or an enzyme. In our experimental design, MWCNTs were

273

used as solid support for immobilization of the antibody. We tried to introduce a simple and

274

easy-to-use separation method that allowed us to identify the reagent bound to MWCNTs.

275

The MWCNTs used here have an outer diameter >50nm and a length in the range of 0.5–2

276

µm. Therefore, we speculated that MWCNTs could be trapped by a membrane with a small

277

pore size, such as a 0.22 µm NC membrane. To avoid non-specific adsorption, the membrane

278

was blocked using 1% BSA solution, which had proved to be an effective pre-treatment in

279

previous tests. Following the incubation step, the mixture of MWCNTs and other reagents

280

was injected onto the 0.22-µm NC membranes. Unbound reagents, including HRP-GEN,

281

GEN, and anti-GEN MAb, were filtered out and the membrane was washed with 5mL PBST

282

to remove the reagents residing in the NC membrane. The color was developed by the

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addition of TMB. The appearance of it was of interest in that blue circles were observed,

284

instead of the expected blue dot or diffuse blue circle and also black particles were found at

285

the center of membrane, which were visible to the naked eye. It is possible that the complex

286

of MWCNTs-PEG-GAM and HRP-GEN was forced to the edge of the NC membrane as a

287

result of the high pressure generated when the mixture of MWCNTs and other reagents was

288

injected onto the NC membrane by syringe. Hence, the blue color was formed at the edge of

289

membrane, resulting in a circular appearance. The color of circles had a non-homogeneous

290

distribution, and gaps even existed in some circles. It was speculated that there were two

291

possible reasons for this phenomenon. First, the diameter of the NC membranes was 1 cm,

292

and the number of MWCNT complexes might not have been sufficient for even distribution

293

in the membranes. Second, the heterogeneity of the NC membranes used might have resulted

294

in uneven diffusion of the solution. As shown in Fig 5, in the absence of GEN, a dark blue

295

circle was clearly visible. At 0.1 ng/mL GEN, the color of the blue circle was lighter and at 1

296

ng/mL GEN the blue color was not visible. When the concentration of GEN was between 0

297

and 0.1 ng/mL, such as 0.05 ng/mL, the color of the circle could not be distinguished from

298

that from the control (absence of GEN) by the naked eye, and LOD was determined as 0.1

299

ng/mL for this method.

300

The method developed here is significantly different than conventional analytical

301

methods based on NC membranes. Firstly, NC membranes are usually adopted as solid

302

supports for the LFA and dot ELISAs, whereby antibodies or antigens are immobilized on the

303

NC membrane using a dispenser. In our method, MWCNTs was used as the support and the

304

NC membrane played the role as a filter to remove unbound reagents from the MWCNTs.

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Secondly, NC membranes which have a high uniformity and appropriate pore size are critical

306

for the development of the LFA and dot ELISAs. Common NC membranes pore sizes are

307

6µm or 8µm, because too small a size will impede the rate of liquid flow. The 0.22-µm NC

308

membranes used in our method are capable of trapping all of the available MWCNTs. Thirdly,

309

for the LFA, the sample application pad, conjugate pad, NC membrane and adsorption pad

310

are assembled on a plastic backing. In our assay format no costly NC modifications are

311

required. Lastly, based on MWCNTs-PEG-GAM, a common analytical method could be

312

developed to detect other targets by adding the corresponding antibody and enzyme conjugate,

313

and could be operated very easily, inexpensively and could be adopted in a variety of

314

laboratory applications.

315 316

3.5 Analysis of milk samples

317

Milk, especially whole milk, contains fat, protein, carbohydrate, minerals and as well as

318

other ingredients, all of which might influence the ability to detect analytes contained within

319

it. To correct for the effect of milk components on the ability to detect GEN, standard curves

320

were constructed by spiking GEN with amounts of milk sample (Fig. 6). When undiluted

321

milk was used, it was found that ODmax decreased significantly and the curve became

322

flattened, which suggested a significant reduction in sensitivity. With increasing dilution,

323

ODmax increased and reached a level equivalent to the ODmax using PBS when either 1:5 or

324

1:10 milk dilutions were adopted. Accordingly, a 1:5 dilution in PBS was employed to

325

prepare milk samples for analysis.

326

Milk samples spiked with GEN standard were evaluated by the quantitative

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centrifugation-based method. Recoveries of GEN in milk ranged from 87.82% to 106.23%

328

(Table 3), demonstrating that the recovery in the proposed method is satisfactory.

329

Then, five milk samples from local markets were analyzed using the protocol referred to

330

above. In the five samples GEN was detected at a range of concentrations from 0.081 to 1.09

331

ng/mL, which were much lower than the established MRL. The results from the quantitative

332

and qualitative methods showed a good correlation(Table 4). According to data above, we

333

suggest that the two immunoassays developed using MWCNTs as support could be effective

334

tools to measure GEN in commercial milk samples.

335 336

3.6 Comparison of methods for GEN based on different solid phases

337

Table 5 shows a summary of the currently published data from quantitative and

338

qualitative GEN immunoassays compared to our observations. It is clear that the

339

immunoassays based on MWCNTs developed here showed higher sensitivity than the

340

previously reported analytical methods. Compared with conventional ELISA, the sensitivity

341

of the new quantitative method decreased slightly, with IC50 of 0.095 ng/mL and 0.195 ng/mL,

342

respectively. It is speculated that the heterogeneity of the MWCNTs led to this result. In

343

heterogeneous immunoassays, an ideal solid support has high binding capacity and

344

homogeneity, such as minimal differences between the holes in the 96-well plate. In our

345

experiment, carboxylated MWCNTs had better dispersion than MWCNTs, but could not be

346

dispersed in aqueous solution stably. In every reaction system, the amount of MWCNTs and

347

antibody fixed on the MWCNTs had some differences, and this heterogeneity likely

348

contributed to the slight decline in sensitivity.

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349 350

3.7 Summary of the quantitative and qualitative assay schemes

351

In summary, the assays can be described as follows. MWCNTs-PEG-GAM, anti-GEN

352

MAb, HRP-GEN and GEN standard were mixed together in solution. The anti-GEN MAb by

353

virtue of its binding to covalently immobilized GAM is captured on the surface of the

354

MWCNT, and it can then capture soluble GEN or HRP-GEN both of which compete for

355

binding to anti-GEN MAb. Therefore, in both assays a loss-of-signal is indicative of

356

increased GEN levels. In the quantitative assay, centrifugation was used to separate the bound

357

reagents from the unbound reagents present in the supernatant and the precipitate was washed

358

to remove non-specifically bound material. In the qualitative assay, unbound reagents were

359

removed using a small pore filter and the HRP trapped on the filter was detected by color

360

generation using a solution of TMB. As in the quantitative assay, the color signal was reduced

361

as the levels of GEN increased.

362

In conclusion, a quantitative assay and a qualitative assay to detect GEN based on

363

MWCNTs as solid support were described in which a simple centrifugation or filtration

364

separation steps were employed. The two methods, especially the qualitative assay, showed

365

high sensitivity and convenience of use without needing a number of tedious steps. Therefore

366

these assays have the potential to become a useful platform for screening residues in foods of

367

animal origin in order to strengthen the safety of animal feed.

368

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ABBREVIATIONS

370

BSA, bovine serum albumin

371

CNT, carbon nanotube

372

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

373

ELISA, enzyme linked immunoadsorbent assay

374

FCA, Freund’s complete adjuvant

375

FIA, Freund’s incomplete adjuvant

376

GAM, goat anti-mouse antibody

377

GEN, gentamicin

378

HPLC, high-performance liquid chromatography

379

HRP, horseradish peroxidase

380

LC-MS, liquid chromatography-mass spectrometry

381

LFA, lateral flow assay

382

LOD, limit of detection

383

MAb monoclonal antibody

384

MES, 2-(N-morpholino)ethanesulfonic acid

385

MNP, magnetic particles

386

MRL, maximum residue limit

387

MWCNT, multi-walled CNT

388

NC, nitrocellulose

389

NH2-PEG-COOH, bifunctional PEG

390

NHS, N-hydroxysuccinimide

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NSB, non-specific binding

392

OVA, ovalbumin

393

PEG, polyethyleneglycol

394

PBS, phosphate buffered saline

395

PBST, phosphate buffered saline with 0.05% Tween20

396

PVDF, polyvinylidene fluoride

397

SWCNT, single-walled CNT

398

TEM, transmission electron microscopy

399

TMB, tetramethylbenzidine (TMB)

Page 20 of 35

400 401

ACKNOWLEDGEMENTS

402

FUNDING SOURCES

403

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

404

(31502118, 31170386, and 31570414), the China Postdoctoral Science Foundation

405

(2013M541606), the Natural Science Fund project of Jiangsu Province (BK20130507), the

406

Scientific Research Funds in Jiangsu University (13JDG016), the Environmental Chemistry

407

and Ecotoxicology State Key Laboratory Fund (KF2014-02) and the Jiangsu Collaborative

408

Innovation Center of Technology and Material of Water Treatment.

409 410

CONFLICT OF INTEREST: The authors declare no competing financial interest.

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Journal of Agricultural and Food Chemistry

REFERENCES

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531 532 533 534 535

FIGURE CAPTIONS

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Figure 1 TEM image of MWCNTs alone and coupled directly or indirectly to GAM. a:

537

untreated MWCNTs; b: carboxylated MWCNTs; c:MWCNTs-GAM;

538

d:MWCNTs-PEG-GAM.

539

Figure 2 Non-specific binding of MWCNTs in the presence or absence of blocking agents

540

Figure 3 Optimization of parameters a: GAM dependence; b: pH dependence; c: time

541

dependence

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Figure 4 GEN standard curve in the centrifugation-based assay

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Figure 5 Representative images for GEN detection by the filtration-based qualitative assay

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Figure 6 GEN standard calibration curves prepared in the presence of different dilutions of

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milk

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TABLES Table 1. Cross-reactivity of MAb to GEN and related compounds Compounds

IC50(ng/mL) )

Cross-reactivity (%)

Gentamicin

0.095

100

Tobramycin

30.8

0.31%

Neomycin

1594.5