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Detection of multiple pathogens in serum using silicaencapsulated nanotags in a SERS-based immunoassay Jing Neng, Yina Li, Ashley J Driscoll, William C Wilson, and Patrick Alfred Johnson J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018
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Journal of Agricultural and Food Chemistry
Detection of multiple pathogens in serum using silica-encapsulated nanotags in a SERS-based immunoassay Jing Nenga, Yina Lib, Ashley J. Driscollb, William C. Wilsonc, Patrick A. Johnsonb*
a
Current address: Department of Food Science and Technology,Zhejiang University of
Technology, Hangzhou, Zhejiang 310014, China. b
Department of Chemical Engineering, University of Wyoming, Laramie, WY 82071,
United States. c
Arthropod-Borne Animal Diseases Research Unit, Center for Grain and Animal Health
Research, USDA/ARS, Manhattan, KS 66502, United States.
*
Corresponding author at: Department of Chemical Engineering, University of
Wyoming, Laramie, WY 82071, USA, Tel.: +1 307 766 6524, E-mail address:
[email protected] (P.A. Johnson).
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Abstract 1
A robust immunoassay based on surface enhanced Raman scattering (SERS) has been
2
developed to simultaneously detect trace quantities of multiple pathogenic antigens from
3
West Nile virus, Rift Valley Fever virus and Yersinia pestis in fetal bovine serum.
4
Antigens were detected by capture with silica-encapsulated nanotags and magnetic
5
nanoparticles conjugated with polyclonal antibodies. The magnetic pull-down resulted in
6
aggregation of the immune complexes and the silica-encapsulated nanotags provided
7
distinct spectra corresponding to each antigen captured. The limit of the detection was ~
8
10 pg/ml in 20% fetal bovine serum, a significant improvement over previous studies in
9
terms of sensitivity, level of multiplexing and medium complexity. This highly sensitive
10
multiplex immunoassay platform provides a promising method to detect various antigens
11
directly in crude serum samples without the tedious process of sample preparation, which
12
is desirable for on-site diagnostic testing and real-time disease monitoring.
13
Keywords
14
Surface-enhanced Raman scattering, multiplex immunoassay, magnetic nanoparticle,
15
West Nile Virus, Rift Valley Fever Virus, Yersinia pestis
16 17 18
INTRODUCTION
19
The development of surface enhanced Raman scattering spectroscopy (SERS) as a
20
detection modality for both portable diagnostic tools and high throughput laboratory
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analysis has garnered significant attention over the past two decades
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SERS include the large numbers of Raman compounds available as reporters for a wide
23
range of laser excitation wavelengths 3, the use of one single excitation wavelength for
24
multiple species
25
fingerprint-like signature profiles for each reporter. With these unique merits enabling
26
high level multiplex analysis 7-9, SERS has been successfully applied to develop complex
27
bioassays, such as multiple DNA detection
28
biomedical diagnosis
29
nanotags are less susceptible to photobleaching and self-quenching
30
laser at 785 nm used in this study can further circumvent background fluorescence issues
31
within biological matrices
32
application of the multiplex SERS-based immunoassays to highly complex biological
33
matrices, such as blood 21, embryos 22 and in vivo tumors 23.
34
To improve the stability of SERS nanotags and the specificity of multiplex SERS
35
bioassays, SERS nanotags with protective shells have been developed to prevent the
36
leaching of Raman reporters and capture biomolecules as well as to prevent cross-talk
37
between two different unshielded SERS nanotags which can lead to false positives 9.
38
Further, these SERS nanotags shield reporter molecules from reacting with contaminants
39
in the sample and prevent the nanotags from aggregating. Several approaches have been
40
used to fabricate the shell-protected SERS nanotags, such as silica encapsulation and
41
polymer coatings
42
biocompatibility, stability, optical transparency and ease of surface modification
43
two independent studies
4-6
24
. The merits of
and above all, the narrow Raman scattering peaks that confer
13-15
10, 11
and multiple protein detection
12
, for
and pathogen screening 16-18. Unlike fluorescent probes, SERS
20
.
19
. The near-infrared
Hence, the intrinsic robustness of SERS extends the
. Silica encapsulation has become widely adopted due to its
26, 27
25
. In
, gold nanoparticles were first incubated with Raman
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reporters before interacting with silane coupling agents, followed by the formation of a
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silica shell via Stöber’s method
46
stabilize gold nanoparticles 29, however, competed with the adsorbed Raman reporters on
47
the gold surface. To address this issue, a layer-by-layer silica encapsulation approach was
48
developed, in which the addition of the polyacrylic acid layer eliminated the competitive
49
adsorption of thiol reagent, and thus improved the surface coverage of Raman reporters
50
20
51
bioassays in complex milieu 24, and the silica-encapsulated nanotags have been applied to
52
various biomedical diagnostics in biological matrices, ranging from detecting tumor cells
53
in whole human blood 21 to multiplexed imaging in living mice 30.
28
. The thiol-polyethylene glycol compound used to
. Overall, the protective shell on the nanotags improves the performance of SERS-based
54 55
The co-infection of multiple pathogens such as bacterial and viral pathogens in
56
agriculturally important animals and crops is not unusual in the same host
57
work, we report a robust and highly sensitive SERS-based immunoassay using magnetic
58
capture of silica-encapsulated SERS nanotags to simultaneously detect two viral antigens
59
and one bacterial antigen, West Nile virus (WNV) envelope (E) protein, Rift Valley fever
60
virus (RVFV) nucleocapsid (N) protein and capsular antigen fraction 1 (F1) from
61
Yersinia pestis in simulated serum samples with a portable Raman instrument. Early
62
detection of pathogenic antigens during infection has great significance in epidemic
63
prevention and therapeutic treatment. In the early stage of viral infection, the exogenous
64
antigen is generally of trace amount, which makes antigen detection within serum
65
challenging. To date, only a few studies have demonstrated simultaneous detection of two
66
or more pathogens in serum or blood with SERS-based technology. In a recent SERS-
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. In this
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based immunoassay study, femtogram detection limits of two antigens were achieved but
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required hours of preparation
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three antigens was recently reported as the first case of using the SERS-based lateral flow
70
assay with a Raman microscope system 34. In comparison, our SERS-based immunoassay
71
employing a colloidal particle system instead of the solid surface system, which
72
improved the reaction kinetics leading to a more efficient detection 35. To our knowledge,
73
this work is the first report of the use of a portable Raman instrument to quantitatively
74
and simultaneously detect three antigens of trace quantities in simulated serum samples in
75
under an hour with no sample preparation.
33
. In another advancement, the simultaneous detection of
76
77
MATERIALS AND METHODS
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Reagents, Materials, and Basic Apparatus. Colloidal gold nanoparticles (60nm,
79
2.6×1010 particles/ml) were purchased from Ted Pella Inc. (Redding, CA) and Fe3O4
80
nanoparticles (~200 nm) were purchased from Nanostructured and Amorphous Materials,
81
Inc. (Houston, TX). Bis-N-succinimidyl-(pentaethylene glycol) ester linker (BS(PEG)12),
82
succinimidyl-[(N-maleimidopropionamido) dodecaethyleneglycol]) ester linker
83
(SM(PEG)12), ammonium hydroxide, Pierce™ protein A/G agarose and the Supersignal®
84
West Pico Chemiluminescent Substrate kit were obtained from Thermo Fisher Scientific
85
(Waltham, MA). HiTrap™ NHS-activated HP columns were ordered from GE
86
Healthcare (Piscataway, NJ). 3-aminopropyltrimethoxysilane (APTMS) (97%), tetraethyl
87
orthosilicate (TEOS) (98%), 3-mercaptopropyltriethoxysilane (MPTES), 3-
88
aminopropyltriethoxysilane (APTES), anhydrous dimethyl sulfoxide (DMSO), laser
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grade Nile blue A perchlorate (NB), Janus Green B (JG), methylene blue hydrate (MB)
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and all other chemicals were from Sigma-Aldrich (St. Louis, MO). Chemical stock
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solutions were made using deionized water (18.2 MΩ cm-1) prepared with Millipore Mill-
92
Q A10 purification system, which were filtered by passage through 0.2 µm Millipore
93
filtration units before use.
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WNV E, RVFV N and F1 antigen/antibody(IgG) reagents. The recombinant His+-
95
tagged WNV E protein and rabbit anti-E polyclonal antibodies have been described
96
previously 36. Rabbit polyclonal antibodies raised against a recombinant His+-tagged
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fusion of RVFV N protein lacking the transmembrane domain were prepared by Drolet et
98
al 37. Yersinia pestis capsular antigen fraction 1 (F1) and antibody were provided by Dr.
99
Gerard Andrews, Department of Veterinary Sciences, University of Wyoming.
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Contaminating anti-E. coli antibodies were removed from serum by total E. coli protein-
101
bound HiTrap™ columns, and the polyclonal antibodies against F1 was then purified
102
from the serum by affinity chromatography with a Protein A/G resin. To evaluate
103
antibody/antigen recognition specificity, replicate Western blots containing three antigens
104
were probed individually with anti-E IgG, anti-N IgG and anti-F IgG, followed by
105
incubation with HRP (horseradish peroxidase)-conjugated goat anti-rabbit IgG and
106
chemiluminescence detection.
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Synthesis of antibody-conjugated magnetic nanoparticles. The antibody-conjugated
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magnetic nanoparticles were synthesized as previously reported 18 with slight
109
modification. First, 1 mg/ml of Fe3O4 nanoparticles in anhydrous DMSO were silanized
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by dropwise addition of APTES to a final concentration of 5% (v/v) 38. The mixture was
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shaken for 15 h at 40°C and an external magnet was then applied to collect Fe3O4
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nanoparticles, followed by several consecutive washings with DMSO to remove unbound
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APTES. Afterwards, 2.5 mg of BS(PEG)12 linker was added to the amine-functionalized
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Fe3O4 nanoparticles redispersed in 1 ml of DMSO. After 6 h incubation at room
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temperature, the above nanoparticle/PEG conjugates were separated from solution by
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magnetic collection and washed several times with phosphate buffered saline (PBS) at pH
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7.5 to remove excess PEG linker and then resuspended in 1 ml PBS. Subsequently, three
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40 µl aliquots of the nanoparticle/PEG conjugates were respectively reacted with 200 µl
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of 0.2 mg/ml anti-E, anti-N and anti-F IgGs, followed by incubation for 6 h at room
120
temperature with gentle shaking, producing nanoparticle/PEG/anti-E,
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nanoparticle/PEG/anti-N and nanoparticle/PEG/anti-F bioconjugates (referred to
122
hereafter as capture magnetic nanoparticles). Finally, the three bioconjugates were
123
washed repeatedly with PBS to remove unconjugated antibody and then resuspended in
124
240 µl of PBS for use in antigen-targeting detection assays.
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Synthesis of silica-encapsulated and antibody-conjugated SERS nanotags. Silica
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encapsulated SERS nanotags were synthesized by a layer-by-layer method 20. First, 20 µl
127
of appropriate concentration of Raman reporters (JG 10-3 M, NB 8 × 10-4 M, MB 2 × 10-4
128
M) was added into 6 ml of colloidal Au suspensions under vigorous stirring and the
129
mixture was equilibrated for 30 min. Next, 6 ml of polyacrylic acid (PAA) adjusted to pH
130
7.0 by 0.1 M NaOH was added dropwise into the mixture under rapid stirring for 3 h. The
131
PAA- encapsulated nanotags were washed three times to remove extra Raman reporters
132
and polymer molecules and then resuspended into water. APTMS was then added to a
133
final concentration of 3 × 10-7 M followed by a 30 minutes equilibration step. A silica
134
shell was subsequently grown by a modified Stöber’s method. Briefly, 17 ml of 2-
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propanol and 200 µl of ammonia hydroxide were added successively with gentle stirring.
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Afterwards, 12 µl of TEOS was added in six portions over a time interval of 1 h and then
137
the mixture was reacted for 12 h. After addition of the TEOS, centrifugation (5000 rpm,
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15 min) was applied to remove free silica nuclei. The collected silica-encapsulated
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nanotags respectively labeled with JG, NB, and MB were resuspended in water and
140
adjusted to a neutral pH.
141
MPTES in ethanol was added to 600 µl aliquots of the silica-encapsulated nanotags to a
142
final concentration of 3 × 10-7 M. The mixture was incubated for 6 h at 37 ºC in a shaker.
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After washing away unreacted MPTES by centrifugation, 0.25 mg of SM(PEG)12
144
dispersed in DMSO was added dropwise and the mixture was incubated for 6 h at room
145
temperature with gentle shaking. Following several washes with PBS to remove excess
146
linker and resuspension in PBS, the PEG-linked silica-encapsulated nanotags were
147
individually reacted with corresponding IgG-enriched antiserum (0.25 mg) for 3 h at
148
room temperature under gentle shaking. Janus Green labeled silica-encapsulated nanotags
149
were functionalized with anti-E IgG, NB-labeled silica-encapsulated nanotags with anti-
150
N IgG and MB-labeled silica-encapsulated nanotags with anti-F IgG. The antibody-
151
functionalized silica-encapsulated nanotags, referred to hereafter as reporter nanotags,
152
were washed several times by centrifugation/resuspension process and the final pellets
153
were resuspended in PBS at a desired concentration for multiplexing.
154
SERS detection of multiple antigen targets by magnetic capture. Multiplex assays
155
were conducted by mixing 45 µl of each reporter nanotag, 45 µl of 20% fetal bovine
156
serum containing dilutions of WNV E, RVFV N and F1 antigen, and 10 µl of each
157
capture magnetic nanoparticle. The ratio of each reporter nanotag and its corresponding
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capture magnetic nanoparticle was 1.1: 1. Triplicate reactions for each concentration of
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antigen were incubated for 1 h at room temperature. The immune complexes were then
160
separated and concentrated from solution using a small external magnet (Ø20 mm × 10
161
mm). The concentrated pellets were interrogated by the laser of an Advantage NIRTM
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Raman spectrometer (60mW, 785nm laser), (DeltaNu, Laramie, WY) fitted with a right
163
angle input optics accessory. Spectroscopic analysis was carried out using a custom
164
Python code in Jupyter Notebook. Raman spectra were recorded in a range of 200-2000
165
cm-1 with baseline off. Each recorded spectrum was an average of five independent data
166
acquisitions (1 s integration time).
167 168
Results and discussion
169
The TOC Figure illustrates the characteristics and mechanism of the immunoassay for
170
target antigen detection using capture magnetic nanoparticles and reporter nanotags.
171
Capture magnetic nanoparticles and reporter nanotags are incubated with a serum sample
172
containing target antigens. During incubation, the polyclonal antibodies conjugated on
173
magnetic nanoparticles and nanotags bind to different epitopes of the same antigen and
174
form immune complexes. These immune complexes are aggregated at the interrogating
175
laser spot by an external magnet source. For multiplex detection, immunoassays are
176
conducted by an assembly of the three capture magnetic nanoparticles and the three
177
corresponding reporter nanotags for WNV E, RVFV N and F1 antigens. The specificity is
178
based on the selection of antibody reagents that do not exhibit antigen cross recognition,
179
the choice of Raman dyes with distinguishable peaks that do not overlap with each other,
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and furthermore, the use of silica-encapsulated Raman nanotags that are impervious to
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matrix and ionic effects, which is especially useful for detection in complex milieu such
182
as serum samples where heterogeneous proteins co-exist.
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The thickness of silica shell plays an important role in the performance of silica-
184
encapsulated nanotags. A silica shell of ideal thickness should restrict massive
185
nanoparticle aggregation, minimize its interference in laser penetration at the same time,
186
and more importantly, it should depend on the diameter of the gold core 29. The thickness
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of the silica shell synthesized on nanotags is approximately 10-15 nm (Figure 1. A),
188
which proved sufficient in blocking both the diffusion of Raman reporters away from the
189
surface and the penetration of external moieties while still maintaining high SERS
190
intensity. During the silica encapsulation, more than one nanotag can be encapsulated
191
together (Figure 1. B), and these clustered nanotags may enhance the Raman signal
192
intensity better than a single encapsulated nanotag 39.
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One major difficulty in multiplex detection is the design of spectrally distinct SERS
194
nanotags with comparable intensities. When the intensities of the three reporters vary too
195
much, the signal of the strongest one can dominate the mixture, rendering it difficult to
196
distinguish and quantify the signal of weaker reporters. By adjusting the concentration of
197
each Raman dye in the silica shell, all three reporter nanotags were able to exhibit
198
distinguishable Raman signals with similar intensity and minimal overlap in the
199
immunoassay for multiplex detection, which is crucial for parallel detection of multiple
200
analytes (Figure 2).
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To evaluate multiplex assay performance in biological matrices, the antigens were spiked
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in fetal bovine serum which was then diluted in PBS to a concentration of 20% (w/v)
203
with antigen concentration from 100 ng/ml to 10 pg/mL. The immunoassay yields intense
204
spectra, as shown in Figure 2. Spectrum A is from the multiplex immunoassay detecting
205
three antigens (10 ng/ml of each) in 20% fetal bovine serum and is characterized by
206
dominant peaks between 400-540 cm-1, 570-695 cm-1, and 1065-1160 cm-1, which are a
207
combination of three signals from methylene blue conjugated nanotags for F1 antigen
208
detection, Nile blue conjugated nanotags for RVFV N antigen detection, and Janus Green
209
labeled nanotags for WNV E antigen detection. Spectrum B is from an immunoassay
210
detecting only WNV E antigen (10 ng/ml), and displays only the characteristic peaks
211
between 1065-1160 cm-1, which are corresponding to the Janus Green labeled SERS
212
nanotags. Spectrum C is from an immunoassay detecting only RVFV N antigen (10
213
ng/ml), and the specific peaks between 570-695 cm-1 are consistent with the Nile blue
214
labeled SERS nanotags. Spectrum D is from an immunoassay detecting only F1 antigen
215
(10 ng/ml), and the distinctive peaks between 400-540 cm-1 match the methylene blue
216
labeled SERS nanotags. Spectra B, C and D confirm that the silica encapsulation prevents
217
cross-talk and increases the specificity of the multiplex immunoassay, for each of these
218
spectra shows only the characteristic peaks of one single reporter labeled SERS nanotag.
219
In the negative control immunoassay, all three antigens were omitted from reporter
220
nanotags and capture magnetic nanoparticles reagents only demonstrate negligible
221
background peaks (spectrum E).
222
To determine the multiplex limit of detection for antigen capture, triplex spectra were
223
acquired for a broad range of F1, RVFV N and WNV E dilutions. As shown in Figure 3,
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a progressive decrease in intensity of characteristic peaks (between 400-540 cm-1, 570-
225
695 cm-1, and 1065-1160 cm-1) is observed as the concentration of the three antigens
226
decreased from 100 ng/ml to 10 pg/ml (A to F). The characteristic peaks in Spectrum F,
227
which were obtained from detecting 10 pg/ml of the three antigens, are still
228
distinguishable compared to Spectrum G, the negative control (Figure 3. inset).
229
Therefore, we conservatively conclude the limit of detection is a minimum of 10 pg/ml
230
(Spectrum F) in 20% fetal bovine serum.
231
To determine the quantitative response of SERS signal intensity to antigen concentration,
232
multiplex spectra were background corrected, and the integration areas under the
233
methylene blue peaks between 400-540 cm-1, Nile blue between 570-695 cm-1 and Janus
234
Green between 1065-1160 cm-1 were independently plotted as a function of their
235
respective antigen concentrations. A log-linear relationship for over three orders of
236
magnitude is found from 10 pg/ml to 10 ng/ml (Figure 4) and linear regression analysis
237
yields an R2 value of 0.9831 for F1 detection, 0.9424 for RVFV N detection and 0.9501
238
for WNV E antigen. The optimal concentration of Raman reporters and the best
239
stoichiometric ratio of reporter nanotags and capture magnetic nanoparticles are currently
240
under investigation.
241
We have demonstrated that a robust, high-level multiplex immunoassay based on SERS
242
can simultaneously detect three antigens in simulated serum samples by magnetic
243
aggregation
244
nanoparticles immune complexes. The limit of detection for WNV E, RVFV N and F1
245
antigen in 20% fetal bovine serum, 10 pg/ml, is 100-fold lower than the single antigen
246
capture assay in 10% serum
of
the
corresponding
40
silica-encapsulated
nanotags/antigen/magnetic
and the duplex antigen detection in PBS buffer
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. In
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addition to the high sensitivity, this SERS-based immunoassay has high specificity and
248
robustness: the protective silica shell not only facilitates immobilization of the Raman
249
reporters on the SERS nanotag surface preventing cross-talk, but also blocks the
250
unfavorable competitive adsorption of the heterogeneous proteins in serum or blood
251
samples. Given that the sample size is of tens of microliters and it demands no
252
complicating operation such as sample preparation, this SERS-based immunoassay can
253
be applied to the onsite diagnostic testing and real-time disease monitoring of
254
agriculturally relevant animals. With little modification, this modality may be adapted for
255
the detection of a wide range of antigens in different biological matrices. Further
256
improvements will focus on expediting assay times, improving the long-term
257
performances of silica-encapsulated nanotags, and developing a portable Raman reader
258
that is specially engineered for magnetic capture of nanotags.
259
260
Acknowledgements
261
The authors thank the financial support from Kansas Bioscience Authority (KBA)
262
(Olathe, KS) and acknowledge the ABADR unit of CGAHR for providing recombinant
263
RVFV N antigens and rabbit anti-N polyclonal antibodies. We also appreciate receiving
264
F1 antigen and antibody reagents from Dr. Gerard Andrews (Department of Veterinary
265
Sciences, University of Wyoming). The contents of this publication are the responsibility
266
of the authors and do not necessarily represent the views of KBA and USDA/ARS.
267
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Figure 1. TEM images of antibody-functionalized silica-encapsulated nanotags: (A)
270
monomer and (B) trimer.
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Figure 2. SERS immunoassays were performed in 20% fetal bovine serum and contained
274
all three reporter nanotags and all three capture magnetic nanoparticles specific for WNV
275
E, RVFV N and F1 antigens. (A) All three antigens: WNV E, RVFV N and F1 (B) WNV
276
E antigen only(C) RVFV N antigen only (D) F1 antigen only and (E) No antigen present
277
(negative control). All immunoassays contained 10 ng/ml of antigen except the negative
278
control. kcts/s = 1000 counts/s.
279 280
Figure 3. SERS spectra for triplex immunoassays of a serial dilution of the F1, RVFV N
281
and WNV E antigens. Stacked spectra correspond to concentrations of (A) 100 ng/ml, (B)
282
10 ng/ml, (C) 1 ng/ml, (D) 0.1 ng/ml, (E) 50 pg/ml, and (F) 10 pg/ml of each antigen.
283
Spectrum G is the negative control without any antigen. Compared to the negative
284
control, the characteristic peaks for a positive detection are still discernable even at
285
concentrations of 10 pg/mL (inset). kcts/s = 1000 counts/s.
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Figure 4. Dose-responsive curves for triplex antigen capture in 20% fetal bovine serum.
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Plots of the log10 concentration of antigen versus signal intensity (area under curve =
290
AUC) of the characteristic peak(s) between (A) 400-540 cm-1 for F1 antigen, (B) 570-695
291
cm-1 for RVFV N antigen, and (C) 1065-1160 cm-1 for WNV E antigen.
292 293
References
294 295 296
1. Driscoll, A. J.; Harpster, M. H.; Johnson, P. A., The development of surface-enhanced Raman scattering as a detection modality for portable in vitro diagnostics: progress and challenges. Physical Chemistry Chemical Physics 2013, 15 (47), 20415-20433.
297 298
2. Laing, S.; Gracie, K.; Faulds, K., Multiplex in vitro detection using SERS. Chemical Society Reviews 2016, 45 (7), 1901-1918.
299 300 301
3. Sun, L.; Yu, C. X.; Irudayaraj, J., Surface-enhanced raman scattering based nonfluorescent probe for multiplex DNA detection. Analytical Chemistry 2007, 79 (11), 3981-3988.
302 303
4. Wang, H. N.; Vo-Dinh, T., Multiplex detection of breast cancer biomarkers using plasmonic molecular sentinel nanoprobes. Nanotechnology 2009, 20 (6), 6.
304 305
5. Kang, T.; Yoo, S. M.; Yoon, I.; Lee, S. Y.; Kim, B., Patterned Multiplex Pathogen DNA Detection by Au Particle-on-Wire SERS Sensor. Nano Letters 2010, 10 (4), 1189-1193.
ACS Paragon Plus Environment
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Page 17 of 20
Journal of Agricultural and Food Chemistry
306 307 308
6. Shen, A. G.; Chen, L. F.; Xie, W.; Hu, J. C.; Zeng, A.; Richards, R.; Hu, J. M., Triplex AuAg-C Core Shell Nanoparticles as a Novel Raman Label. Advanced Functional Materials 2010, 20 (6), 969-975.
309 310 311
7. Graham, D.; Mallinder, B. J.; Whitcombe, D.; Watson, N. D.; Smith, W. E., Simple multiplex genotyping by surface-enhanced resonance Raman scattering. Analytical Chemistry 2002, 74 (5), 1069-+.
312 313
8. Faulds, K.; Smith, W. E.; Graham, D., Evaluation of surface-enhanced resonance Raman scattering for quantitative DNA analysis. Analytical Chemistry 2004, 76 (2), 412-417.
314 315 316
9. Porter, M. D.; Lipert, R. J.; Siperko, L. M.; Wang, G.; Narayanana, R., SERS as a bioassay platform: fundamentals, design, and applications. Chemical Society Reviews 2008, 37 (5), 1001-1011.
317 318
10. Li, Y. G.; Cu, Y. T. H.; Luo, D., Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes. Nature Biotechnology 2005, 23 (7), 885-889.
319 320 321
11. Stokes, R. J.; Macaskill, A.; Lundahl, P. J.; Smith, W. E.; Faulds, K.; Graham, D., Quantitative enhanced Raman scattering of labeled DNA from gold and silver nanoparticles. Small 2007, 3 (9), 1593-1601.
322 323 324 325
12. Jun, B. H.; Noh, M. S.; Kim, J.; Kim, G.; Kang, H.; Kim, M. S.; Seo, Y. T.; Baek, J.; Kim, J. H.; Park, J.; Kim, S.; Kim, Y. K.; Hyeon, T.; Cho, M. H.; Jeong, D. H.; Lee, Y. S., Multifunctional Silver-Embedded Magnetic Nanoparticles as SERS Nanoprobes and Their Applications. Small 2010, 6 (1), 119-125.
326 327 328
13. Maiti, K. K.; Samanta, A.; Vendrell, M.; Soh, K. S.; Olivo, M.; Chang, Y. T., Multiplex cancer cell detection by SERS nanotags with cyanine and triphenylmethine Raman reporters. Chemical Communications 2011, 47 (12), 3514-3516.
329 330 331
14. Dinish, U. S.; Balasundaram, G.; Chang, Y. T.; Olivo, M., Actively Targeted In Vivo Multiplex Detection of Intrinsic Cancer Biomarkers Using Biocompatible SERS Nanotags. Scientific Reports 2014, 4, 7.
332 333 334
15. Lee, S.; Chon, H.; Lee, J.; Ko, J.; Chung, B. H.; Lim, D. W.; Choo, J., Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging. Biosensors & Bioelectronics 2014, 51, 238-243.
335 336 337
16. Cam, D.; Keseroglu, K.; Kahraman, M.; Sahin, F.; Culha, M., Multiplex identification of bacteria in bacterial mixtures with surface-enhanced Raman scattering. Journal of Raman Spectroscopy 2010, 41 (5), 484-489.
338 339
17. Ravindranath, S. P.; Wang, Y. L.; Irudayaraj, J., SERS driven cross-platform based multiplex pathogen detection. Sensors and Actuators B-Chemical 2011, 152 (2), 183-190.
340 341 342
18. Neng, J.; Harpster, M. H.; Wilson, W. C.; Johnson, P. A., Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles. Biosensors & Bioelectronics 2013, 41, 316-321.
343 344
19. Sharma, B.; Frontiera, R. R.; Henry, A. I.; Ringe, E.; Van Duyne, R. P., SERS: Materials, applications, and the future. Materials Today 2012, 15 (1-2), 16-25.
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Page 18 of 20
345 346 347
20. Huang, J.; Kim, K. H.; Choi, N.; Chon, H.; Lee, S.; Choo, J., Preparation of SilicaEncapsulated Hollow Gold Nanosphere Tags Using Layer-by-Layer Method for Multiplex Surface-Enhanced Raman Scattering Detection. Langmuir 2011, 27 (16), 10228-10233.
348 349 350
21. Sha, M. Y.; Xu, H. X.; Natan, M. J.; Cromer, R., Surface-Enhanced Raman Scattering Tags for Rapid and Homogeneous Detection of Circulating Tumor Cells in the Presence of Human Whole Blood. Journal of the American Chemical Society 2008, 130 (51), 17214-+.
351 352 353
22. Wang, Y. L.; Seebald, J. L.; Szeto, D. P.; Irudayaraj, J., Biocompatibility and Biodistribution of Surface-Enhanced Raman Scattering Nanoprobes in Zebrafish Embryos: In vivo and Multiplex Imaging. Acs Nano 2010, 4 (7), 4039-4053.
354 355 356
23. Qian, X. M.; Peng, X. H.; Ansari, D. O.; Yin-Goen, Q.; Chen, G. Z.; Shin, D. M.; Yang, L.; Young, A. N.; Wang, M. D.; Nie, S. M., In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nature Biotechnology 2008, 26 (1), 83-90.
357 358 359
24. Doering, W. E.; Piotti, M. E.; Natan, M. J.; Freeman, R. G., SERS as a foundation for nanoscale, optically detected biological labels. Advanced Materials 2007, 19 (20), 31003108.
360 361 362 363
25. Schiestel, T.; Brunner, H.; Tovar, G. E. M., Controlled surface functionalization of silica nanospheres by covalent conjugation reactions and preparation of high density streptavidin nanoparticles. Journal of Nanoscience and Nanotechnology 2004, 4 (5), 504511.
364 365
26. Doering, W. E.; Nie, S. M., Spectroscopic tags using dye-embedded nanoparticles and surface-enhanced Raman scattering. Analytical Chemistry 2003, 75 (22), 6171-6176.
366 367 368
27. Mulvaney, S. P.; Musick, M. D.; Keating, C. D.; Natan, M. J., Glass-coated, analytetagged nanoparticles: A new tagging system based on detection with surface-enhanced Raman scattering. Langmuir 2003, 19 (11), 4784-4790.
369 370 371
28. Stober, W.; Fink, A.; Bohn, E., CONTROLLED GROWTH OF MONODISPERSE SILICA SPHERES IN MICRON SIZE RANGE. Journal of Colloid and Interface Science 1968, 26 (1), 62&.
372 373 374 375
29. Fernandez-Lopez, C.; Mateo-Mateo, C.; Alvarez-Puebla, R. A.; Perez-Juste, J.; Pastoriza-Santos, I.; Liz-Marzan, L. M., Highly Controlled Silica Coating of PEG-Capped Metal Nanoparticles and Preparation of SERS-Encoded Particles. Langmuir 2009, 25 (24), 1389413899.
376 377 378 379
30. Zavaleta, C. L.; Smith, B. R.; Walton, I.; Doering, W.; Davis, G.; Shojaei, B.; Natan, M. J.; Gambhir, S. S., Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy. Proceedings of the National Academy of Sciences of the United States of America 2009, 106 (32), 13511-13516.
380 381
31. Moore, D. M.; Jaykus, L., Virus–Bacteria Interactions: Implications and Potential for the Applied and Agricultural Sciences. Viruses 2018, 10.
382 383 384
32. Adjou Moumouni, P. F.; Aplogan, G. L.; Katahira, H.; Gao, Y.; Guo, H.; Efstratiou, A.; Jirapattharasate, C.; Wang, G.; Liu, M.; Ringo, A. E.; Umemiya-Shirafuji, R.; Suzuki, H.; Xuan, X., Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens
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Page 19 of 20
Journal of Agricultural and Food Chemistry
385 386
in cattle from Rhipicephalus microplus-invaded and non-invaded areas of Benin. Ticks and Tick-borne Diseases 2018, 9 (3), 450-464.
387 388 389 390
33. Wang, Z.; Yang, H.; Wang, M.; Petti, L.; Jiang, T.; Jia, Z.; Xie, S.; Zhou, J., SERS-based multiplex immunoassay of tumor markers using double SiO 2 @Ag immune probes and gold-film hemisphere array immune substrate. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2018, 546, 48-58.
391 392
34. Zhang, D.; Huang, L.; Liu, B.; Ni, H.; Sun, L.; Su, E.; Chen, H.; Gu, Z.; Zhao, X., Quantitative and ultrasensitive detection of multiplex cardiac biomarkers in
393 394
lateral fl ow assay with core-shell SERS nanotags. Biosensors and Bioelectronics, 2018; Vol. 106, pp 204-211.
395 396 397
35. Driscoll, A. J.; Johnson, P. A., Numerical modeling of analyte diffusion and adsorption behavior in microparticle and nanoparticle based biosensors. Chemical Engineering Science 2018, 184, 141-148.
398 399 400 401
36. Neng, J.; Harpster, M. H.; Zhang, H.; Mecham, J. O.; Wilson, W. C.; Johnson, P. A., A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G. Biosensors & Bioelectronics 2010, 26 (3), 1009-1015.
402 403 404 405
37. Drolet, B. S.; Weingartl, H. M.; Jiang, J. Y.; Neufeld, J.; Marszal, P.; Lindsay, R.; Miller, M. M.; Czub, M.; Wilson, W. C., Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories. Journal of Virological Methods 2012, 179 (2), 373-382.
406 407 408
38. Cao, H. N.; He, J.; Deng, L.; Gao, X. Q., Fabrication of cyclodextrin-functionalized superparamagnetic Fe3O4/amino-silane core-shell nanoparticles via layer-by-layer method. Applied Surface Science 2009, 255 (18), 7974-7980.
409 410 411
39. Salehi, M.; Schneider, L.; Strobel, P.; Marx, A.; Packeisen, J.; Schlucker, S., Two-color SERS microscopy for protein co-localization in prostate tissue with primary antibodyprotein A/G-gold nanocluster conjugates. Nanoscale 2014, 6 (4), 2361-2367.
412 413 414 415
40. Wang, G. F.; Lipert, R. J.; Jain, M.; Kaur, S.; Chakraboty, S.; Torres, M. P.; Batra, S. K.; Brand, R. E.; Porter, M. D., Detection of the Potential Pancreatic Cancer Marker MUC4 in Serum Using Surface-Enhanced Raman Scattering. Analytical Chemistry 2011, 83 (7), 25542561.
416 417 418
41. Chon, H.; Lim, C.; Ha, S. M.; Ahn, Y.; Lee, E. K.; Chang, S. I.; Seong, G. H.; Choo, J., OnChip Immunoassay Using Surface-Enhanced Raman Scattering of Hollow Gold Nanospheres. Analytical Chemistry 2010, 82 (12), 5290-5295.
419 420 421 422
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TOC Graphic
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Schematic of antigen detection in the immunoassay using magnetic nanoparticles and silica-encapsulated SERS nanotags.
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Table of Contents Categories 1. Analytical Methods 2. Food Safety and Toxicology All Figures in color format.
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