Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
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
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 free 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 accessible to all readers and 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.
Journal of Agricultural and Food Chemistry 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 35
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
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
Page 2 of 35
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
2
ACS Paragon Plus Environment
Page 3 of 35
Journal of Agricultural and Food Chemistry
19
1. INTRODUCTION
20
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
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 35
41
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
56
drug-delivery carriers24, gene delivery systems25 and immunodetection methods26. In
57
analytical processes, because of their advantage of having a large surface area, CNTs can be
58
used as solid supports to carry proteins, nucleic acids, and drugs either through covalent or
59
non-covalent binding. Currently CNTs have principally been used in three fields: a)
60
electrochemical biosensors: CNTs can mediate electron-transfer reactions between
61
electroactive species and can thus be developed as electrical sensors19,27; b) signal
62
amplification:CNTs can be linked with enzymes or agents, such as other nanoparticles, by
4
ACS Paragon Plus Environment
Page 5 of 35
Journal of Agricultural and Food Chemistry
63
exploiting the high absorption capacity of CNTs26,28;or c) as a versatile label: CNTs can be
64
used as signaling molecules replacing gold nanoparticles in the lateral flow assay, producing
65
the characteristic black bands for visual detection20,29 However, CNT based bioassays suffer
66
from the requirement for numerous, tedious steps, and a requirement for sophisticated
67
instrumentation.
68
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
73
2.1. Reagents and materials
74
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
5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
85
Page 6 of 35
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
97
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
102
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.
6
ACS Paragon Plus Environment
Page 7 of 35
Journal of Agricultural and Food Chemistry
107 108
2.4 Indirect competitive ELISA for GEN
109
An indirect competition ELISA format was utilized to measure GEN binding and
110
cross-reactivity to related compounds. First, 96-well plates were coated with OVA-GEN
111
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
113
37 °C for 2 h. Then, 50 µL GEN standards, followed by 50 µL anti-GEN antibody, was added
114
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
121
To obtain carboxylated MWCNTs, MWCNTs were treated with a 3:1 (v/v) mixture of
122
sulfuric and nitric acids at 65°C for 4 h. The carboxylated MWCNTs were filtered and
123
washed repeatedly with distilled water until the pH of water was neutral. The product was
124
dried at 50°C
125
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
127
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
7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 35
129
washed with MES (50 mmol/L, pH 6.1) to remove unconjugated EDC or NHS. After washing
130
twice, the precipitate was resuspended in 1 mL MES (50 mmol/L, pH 6.1) and 400 µg GAM
131
was added. The mixture was stirred overnight at 4°C. The conjugated MWCNTs-GAM was
132
washed three times using PBS (0.01 mol/L, pH 7.2) and stored in 1 mL PBS at 4°C.
133
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
138
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
142
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
149
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
8
ACS Paragon Plus Environment
Page 9 of 35
Journal of Agricultural and Food Chemistry
151
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
156
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,
159
differing amounts of GAM (50–1600 µg) were mixed with MWCNTs-PEG (1 mg). Then,
160
anti-GEN
161
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
163
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.
172
9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
173
Page 10 of 35
2.9 Analysis of commercially available milk
174
Five samples of commercially available milk were obtained from local markets
175
(Zhenjiang, China). To reduce any potential interference of milk ingredients, samples were
176
serially diluted with PBS before analysis.
177 178
3. RESULTS AND DISCUSSION
179
3.1 Characterization of anti-GEN MAbs
180
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
182
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.
185
The cross-reactivity of 2D12 with structural analogs of GEN was examined in order to
186
understand its selectivity (Table 1). The antibody 2D12 showed only weak cross reactivity
187
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
192
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
10
ACS Paragon Plus Environment
Page 11 of 35
195
Journal of Agricultural and Food Chemistry
ng/mL.
196 197
3.2 Characterization of MWCNTs and covalently immobilized antibodies
198
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
11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 12 of 35
217
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
12
ACS Paragon Plus Environment
Page 13 of 35
Journal of Agricultural and Food Chemistry
239
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
13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
261
Page 14 of 35
7.4. Based on these data the optimum pH was chosen to be 7.4.
262
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
14
ACS Paragon Plus Environment
Page 15 of 35
Journal of Agricultural and Food Chemistry
283
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.
15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 16 of 35
305
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
16
ACS Paragon Plus Environment
Page 17 of 35
Journal of Agricultural and Food Chemistry
327
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.
17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 18 of 35
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
18
ACS Paragon Plus Environment
Page 19 of 35
Journal of Agricultural and Food Chemistry
369
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
19
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
391
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.
411
20
ACS Paragon Plus Environment
Page 21 of 35
412
Journal of Agricultural and Food Chemistry
REFERENCES
413
1. Chen, Y.; Shang, Y.; Li, X.; Wu, X.; Xiao, X. Development of an enzyme-linked
414
immunoassay for the detection of gentamicin in swine tissues. Food Chem. 2008,
415
108, 304-309
416
2. Clark, C. Clinical uses of gentamicin. Mod. Vet. Pract. 1977, 58, 751-754
417
3. COMMISSION REGULATION (EU) No 37/2010 of 22 December 2009 on
418
pharmacologically active substances and their classification regarding maximum
419
residue limits in foodstuffs of animal origin.
420
(http://ec.europa.eu/health/files/eudralex/vol-5/reg_2010_37/reg_2010_37_en.pdf)
421
4. Al-Amoud, A.; Clark, B.; Chrystyn, H. Determination of gentamicin in urine
422
samples after inhalation by reversed-phase high-performance liquid
423
chromatography using pre-column derivatisation with o-phthalaldehyde. J.
424
Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2002, 769, 89-95
425
5. Kaufmann, A.; Maden, K. Determination of 11 aminoglycosides in meat and liver
426
by liquid chromatography with tandem mass spectrometry. J. AOAC Int. 2005, 88,
427
1118-1125
428
6. Posyniak, A.; Zmudzki, J.; Niedzielska, J. Sample preparation for residue
429
determination of gentamicin and neomycin by liquid chromatography. J.
430
Chromatogr. A 2001, 914, 59-66
431
7. Lecaroz, C.; Campanero, M.; Gamazo, C.; Blanco-Prieto, M. Determination of
432
gentamicin in different matrices by a new sensitive high-performance liquid
433
chromatography-mass spectrometric method. J. Antimicrob. Chemother. 2006, 58,
21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
434 435 436 437 438 439
Page 22 of 35
557-563 8. Rosner, A.; Aviv, H. Gentamicin bioautography assay vs. the microbiological disk test. J. Antibiot. (Tokyo) 1980, 33, 600-603 9. Wei, M.; Wang, B.; Juan, L.; Chuan-Lai, X. Development of enzyme-linked immunoassay for detection of gentamicin. Food Sci. 2009, 30, 242-244 10. Jin,
Y.; Jang,
J.; Han,
C.; Lee, M. Development of ELISA
and
440
immunochromatographic assay for the detection of gentamicin. J. Agric. Food
441
Chem. 2005, 53, 7639-7643
442
11. Peng, C.; Li, Z.; Zhu, Y.; Chen, W.; Yuan, Y.; Liu, L.; Li, Q.; Xu, D.; Qiao, R.;
443
Wang, L.; Zhu, S.; Jin, Z.; Xu, C. Simultaneous and sensitive determination of
444
multiplex chemical residues based on multicolor quantum dot probes. Biosens.
445
Bioelectron. 2009, 24, 3657-3662
446
12. Kim, S.; Lim, H. Chemiluminescence immunoassay using magnetic nanoparticles
447
with targeted inhibition for the determination of ochratoxin A. Talanta 2015, 140,
448
183-188
449
13. Sun, Q.; Zhao, G.; Dou, W. An optical and rapid sandwich immunoassay method
450
for detection of Salmonella pullorum and Salmonella gallinarum based on
451
immune blue silica nanoparticles and magnetic nanoparticles. Sensor. Actuator. B
452
Chem. 2016, 226, 69-75
453
14. Liu, X.; Hu, Y.; Zheng, S.; Liu, Y.; He, Z.; Luo, F. Surface plasmon resonance
454
immunosensor for fast, highly sensitive, and in situ detection of the magnetic
455
nanoparticles-enriched Salmonella enteritidis. Sensor. Actuator. B Chem. 2016,
22
ACS Paragon Plus Environment
Page 23 of 35
456 457 458
Journal of Agricultural and Food Chemistry
230, 191-8 15. Dzantiev, B.; Byzova, N.; Urusov, A.; & Zherdev, A. Immunochromatographic methods in food analysis. TrAC, Trends Anal. Chem. 2014, 55, 81-93
459
16. Ngom, B.; GuY.; Wang, X.; Bi, D. Development and application of lateral flow
460
test strip technology for detection of infectious agents and chemical contaminants:
461
a review. Anal. Bioanal. Chem. 2010, 397, 1113-1135
462 463
17. Wang, S.; Wei, Y.; Jin, H.; Li C.; Du, H. A 96-well plate based Dot-ELISA array for simultaneous detection of multi-drugs. Anal. Lett. 2009, 42, 2807-2819
464
18. Yu, X.; Munge, B.; Patel, V.; Jensen, G.; Bhirde, A.; Gong, J.; Kim, S.; Gillespie,
465
J.; Gutkind, J.; Papadimitrakopoulos, F.; Rusling, J. Carbon nanotube
466
amplification strategies for highly sensitive immunodetection of cancer
467
biomarkers J. Am. Chem. Soc. 2006, 128, 11199-11205
468 469
19. Pumera, M.; Sánchez, S.; Ichinose, I.; Tang, J. Electrochemical nanobiosensors. Sensor. Actuator. B Chem. 2007 123, 1195-11205
470
20. Posthuma-Trumpie, G.; Wichers, J.; Koets, M.; Berendsen, L.; van Amerongen, A.
471
Amorphous carbon nanoparticles: a versatile label for rapid diagnostic
472
(immuno)assays. Anal. Bioanal. Chem. 2012, 402, 593-600
473
21. Iijima, S. Helical microtubules of graphitic carbon. Nature 1991, 354, 56-58
474
22. Iijima, S. Carbon nanotubes: Past, present, and future. Phys. B Condens. Matter
475 476 477
2002, 323, 1-5 23. Liu, Z.; Tabakman, S.; Welsher, K.; Dai, H. Carbon in biology and medicine: in vitro and in vivo detection, imaging and drug delivery. Nano Res. 2009, 2, 85-120
23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 24 of 35
478
24. Meng, L.; Zhang, X.; Lu, Q.; Fei, Z.; Dyson, P. Single walled carbon nanotubes as
479
drug delivery vehicles: Targeting doxorubicin to tumors. Biomaterials 2012, 33,
480
1689-1698
481 482
25. Abu-Salah, K.; Ansari, A.; Alrokayan, S. DNA-based applications in nanobiotechnology. J. Biomed. Biotechnol. 2010, 2010,715295-715295
483
26. Yu, X.; Munge, B.; Patel, V.; Jensen, G.; Bhirde, A.; Gong, J.; Kim, S.; Gillespie,
484
J.; Gutkind, J.; Papadimitrakopoulos, F.; Rusling, J. Carbon nanotube
485
amplification strategies for highly sensitive immunodetection of cancer
486
biomarkers J. Am. Chem. Soc. 2006, 128, 11199-11205
487
27. Miodek, A.; Mejri, N.; Gomgnimbou, M.; Sola, C.; Korri-Youssoufi, H. E-DNA
488
sensor of Mycobacterium tuberculosis based on electrochemical assembly of
489
nanomaterials (MWCNTs/PPy/PAMAM). Anal. Chem. 2015, 87, 9257-9264.
490
28. Zhang, L.; Chen, J.; Wang, Y.; Lei Yu, Wang, J.; Peng, H.; Zhu, J. Improved
491
enzyme immobilization for enhanced bioelectrocatalytic activity of choline sensor
492
and acetylcholine sensor. Sens. Actuators, B, 2014, 193, 904-910.
493
29. Qiu, W.; Hui, X.; Takalkar, S.; Liu, B.; Zheng, Y.; Guo, Z.; Baloda, M.; Baryeh,
494
K.; Liu, G. Carbon nanotube-based lateral flow biosensor for sensitive and rapid
495
detection of DNA sequence. Biosens. Bioelectron. 2015, 64, 367-372.
496
30. Zeng, K.; Yang, T.; Zhong, P.; Zhou, S.; Qu, L.; He, J.; Jiang, Z. Development of
497
an indirect competitive immunoassay for parathion in vegetables. Food Chem.
498
2007, 102, 1076-1082
499
31. Shalev, M., Kandasamy, J.; Skalka, N.; Belakhov, V.; Rosin-Arbesfeld, R.;
24
ACS Paragon Plus Environment
Page 25 of 35
Journal of Agricultural and Food Chemistry
500
Baasov, T. Development of generic immunoassay for the detection of a series of
501
aminoglycosides with 6'-OH group for the treatment of genetic diseases in
502
biological samples. J. Pharm. Biomed. Anal. 2013, 75, 33-40
503
32. Wang, J.; Cheng, M.; Zhang, Z.; Guo, L.; Liu, Q.; Jiang, G. An antibody-graphene
504
oxide nanoribbon conjugate as a surface enhanced laser desorption/ionization
505
probe with high sensitivity and selectivity. Chem. Commun. (Camb) 2015, 51,
506
4619-4622
507
33. Jeong, B.; Akter, R.; Han, O.; Rhee, C.; Rahman, M. Increased electrocatalyzed
508
performance through dendrimer-encapsulated gold nanoparticles and carbon
509
nanotube-assisted multiple bienzymatic labels: highly sensitive electrochemical
510
immunosensor for protein detection. Anal. Chem. 2013, 85, 1784-1791
511 512
34. Krska, R.; Molinelli, A. Rapid test strips for analysis of mycotoxins in food and feed. Anal. Bioanal. Chem. 2009, 393, 67-71
513
35. Pappas, M.; Hajkowski, R.; Hockmeyer, W. Dot enzyme-linked immunosorbent
514
assay (Dot-ELISA): a micro technique for the rapid diagnosis of visceral
515
leishmaniasis. J. Immunol. Methods 1983, 64, 205-214
516
36. Beloglazova, N.; Shmelin, P.; Eremin, S. Sensitive immunochemical approaches
517
for quantitative (FPIA) and qualitative (lateral flow tests) determination of
518
gentamicin in milk. Talanta 2016, 149, 217-224
519
37. Yang, H.; Zhu, Q.; Qu, H.; Chen, X.; Ding, M.; Xu, G. Flow injection
520
fluorescence immunoassay for gentamicin using sol-gel-derived mesoporous
521
biomaterial. Anal. Biochem. 2002, 308, 71–76
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 26 of 35
522
38. Haasnoot, W., Cazemier, G.; Koets, M.; van Amerongen, A. Single biosensor
523
immunoassay for the detection of five aminoglycosides in reconstituted skimmed
524
milk. Anal. Chim. Acta 2003, 488, 53-60.
525
39. Zhu, Y.; Qu, C.; Kuang, H.; Xu, L.; Liu, L.; Hua, Y.; Wang, L Xu, C. Simple,
526
rapid and sensitive detection of antibiotics based on the side-by-side assembly of
527
gold nanorod probes. Biosens. Bioelectron. 2011, 26, 4387-4392
528
40. Es, R.; Setford, S.; Blankwater, Y.; Meijer, D. Detection of gentamicin in milk by
529
immunoassay and flow injection analysis with electrochemical measurement. Anal.
530
Chim. Acta 2001, 429, 37-47
531 532 533 534 535
FIGURE CAPTIONS
536
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
542
Figure 4 GEN standard curve in the centrifugation-based assay
543
Figure 5 Representative images for GEN detection by the filtration-based qualitative assay
26
ACS Paragon Plus Environment
Page 27 of 35
Journal of Agricultural and Food Chemistry
544
Figure 6 GEN standard calibration curves prepared in the presence of different dilutions of
545
milk
27
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 28 of 35
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